T-cell selective interleukin-4 agonists

ABSTRACT

This invention realizes a less toxic IL-4 mutant that allows greater therapeutic use of interleukin 4. Further, the invention is directed to IL-4 muteins having single and double mutations represented by the designators R121E and T13D/R121E, numbered in accordance with wild type IL-4 (His=1). The invention also includes polynucleotides coding for the muteins of the invention, vectors containing the polynucleotides, transformed host cells, pharmaceutical compositions comprising the muteins, and therapeutic methods of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/298,374, filed Apr. 23, 1999, which was a continuation-in-part ofU.S. application Ser. No. 08/874,697, filed Jun. 13, 1997, which claimsthe benefit of U.S. Provisional Application Nos. 60/036,746, filed Jan.27, 1997, and 60/019,748, filed Jun. 14, 1996.

BACKGROUND

[0002] 1. Field of the Invention

[0003] The invention is generally related to the fields of pharmacologyand immunology. More specifically, the invention is directed to novelcompositions of matter for selectively activating T cells, and havingreduced activation of Endothelial cells or fibroblasts. The novelcompositions include variants of the cytokine family, and in particularhuman Interleukin-4 (IL-4).

[0004] 2. Description of Related Art

[0005] Interleukin 4 (IL-4) is a pleiotropic cytokine, having activitieson cells of the immune system, endothelium, and those of fibroblasticnature. Reported in vitro effects of IL-4 administration includeproliferation of B cells, immunoglobulin class switching in B cells. InT cells, IL-4 stimulates T cell proliferation after preactivation withmitogens and down-regulates IFN-γ production. In monocytes, IL-4 inducesclass II MHC molecules expression, release of lipopolysaccharide-inducedtPA, and CD23 expression. In Endothelial cells (EC), IL4 inducesexpression of VCAM-1 and IL-6 release, and decreases ICAM-1 expression.(Maher, D W, et al., Human Interleukin-4: An Immunomodulator withPotential Therapeutic Applications, Progress in Growth Factor Research,3:43-56 (1991)).

[0006] Because of its ability to stimulate proliferation of T cellsactivated by exposure to IL-2, IL-4 therapy has been pursued. Forinstance, IL-4 has demonstrated anti-neoplastic activity in animalmodels of renal carcinomas, and has induced tumor regression in mice(Bosco, M., et al., Low Doses of IL-4 Injected Perilymphatically inTumor-bearing Mice Inhibit the Growth of Poorly and ApparentlyNonimmunogenic Tumors and Induce a Tumor Specific Immune Memory, J.Immunol., 145:3136-43 (1990)). However, its toxicity limits dosage inhumans (Margolin, K., et al., Phase II Studies of Human RecombinantInterleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J.Immunotherapy, 15:147-153 (1994)).

[0007] Because of its immunoregulatory activity, a number of clinicalapplications are suggested for IL-4. Among these clinical applicationsare disorders caused by imbalances of the immune system, particularlythose caused by imbalances of T helper (Th) cell responses to antigen.These diseases include certain autoimmune diseases, rheumatic diseases,dermatological diseases, and infectious diseases. A large body ofexperimental work has established that Th cells fall into two broadclasses, designated Th1 and Th2 (Mosmann, T. R., Cherwinski, H., Bond,M. W., Giedlin, M. A. and Coffman, R. L., Two types of murine helper Tcell clone. I. Definition according to profiles of lymphokine activitiesand secreted proteins, J. Immunol., 136:2348-2357 (1986); Mosmann, T.R., Cytokines, differentiation and functions of subsets of CD4 and CD8 Tcells, Behring Inst. Mitt., 1-6 (1995)). These T cell classes aredefined by the cytokines they express: Th1 cells make IL-2, INF-γ, andTNF-α, while Th2 cells make IL-4 and IL-5. Th1 and Th2 cells are formedfrom naive CD4+ T cells. Differentiation into Th1 or Th2 subsets dependson the cytokine present during antigen stimulation: IFN-γ and IL-12direct differentiation of naive cells to the Th1 phenotype, while IL-4directs differentiation to the Th2 phenotype. While the Th1 and Th2subsets may represent extremes along a continuum of Th cell phenotypes(for example, Th0 cells, which express low levels of both INF-γ andIL-4, have been described), this classification nevertheless is themajor paradigm in the field of immunology for describing the characterof the immune response.

[0008] It has been observed that certain organ-specific autoimmunediseases are associated with a predominantly Th1 T cell response againstautoantigen (Liblau R S; Singer S M; McDevitt H O, Th1 and Th2 CD4⁺ Tcells in the pathogenesis of organ-specific autoimmune diseases,Immunol. Today, 16:34-38 (1995)). One such autoimmune disease isinsulin-dependent diabetes (IDDM), a disorder characterized by Tcell-mediated destruction of pancreatic β cells. Several lines ofevidence suggest that Th1-type cells are primarily responsible for thepancreatic β cell destruction (reviewed in Tisch, R. et al., Review:Insulin-dependent Diabetes Mellitus, Cell, 85:291-297 (1996)).Administration of IL-4 to NOD mice, which serves as an animal model ofIDDM, down-regulates the Th1 cell population and significantly delaysthe onset of diabetes (Rapoport, et al., IL-4 Reverses T cellProliferation Unresponsiveness and Prevents the Onset of Diabetes in NODMice, J. Exp. Med., 178:87-99 (1993)). Another such autoimmune diseaseis multiple sclerosis (MS), a disease which is characterized by anautoimmune attack upon the myelin sheath surrounding nerve cells.Studies in humans with MS have demonstrated that exacerbation of MS isassociated with the presence of autoantigen-specific Th1 and Th0 cellsand that remission is associated with the presence ofautoantigen-specific Th2 and Th0 cells (Correale, J. et al., Patterns ofcytokine secretion by autoreactive proteolipid protein-specific T cellclones during the course of multiple sclerosis, J. Immunol.,154:2959-2968 (1995)). Mice with experimental autoimmuneencephalomyelitis (EAE), an animal model for MS, also exhibit the Th1cell polarization (Cua, D J, Hinton, D R, and Stohlman, S A, J.Immunol., 155:4052-4059 (1995)). Indirect evidence from a study in theEAE model suggests that IL-4 plays a critical role in diseaseattenuation resulting from treatment with a tolerogenic peptide (Brocke,S. et al. Treatment of experimental encephalomyelitis with a peptideanalogue of myelin basic protein, Nature, 379:343-346 (1996)).

[0009] Other autoimmune diseases such as Rheumatoid Arthritis (RA) arealso targets for IL-4 based therapies. Animal models of RA have shown adisequilibrium of cell profiles tilting towards Th1 cells, and in micethat overexpress TNF-α, anti-TNF-α antibodies have demonstrated diseaseattenuation, suggesting that IL-4 therapies that result indown-regulation of Th1 cell populations may have an anti-TNF-α effectalso. (See Feldmann, M., et al., Review: Rheumatoid Arthritis, Cell,85:307-310 (1996)).

[0010] Psoriasis vulgaris is a chronic dermatologic disordercharacterized by infiltration of affected skin with monocytes and Tcells. Several reports indicate that psoriatic skin lesional T cells andPBL are predominantly of the Th1 phenotype (Uyemura K; Yamamura M;Fivenson D F; Modlin R L; Nickoloff B J, The cytokine network inlesional and lesion-free psoriatic skin is characterized by a T-helpertype 1 cell-mediated response, J Invest Dermatol., 101:701-705 (1993);Schlaak J F; Buslau M; Jochum W; Hermann E; Girndt M; Gallati H; Meyerzum Buschenfelde K H; Fleischer B, T cells involved in psoriasisvulgaris belong to the Th1 subset, J Invest Dermatol, 102:145-149(1994)). Furthermore, monomethylfumarate, a drug which has been reportedto be of clinical benefit to patients with psoriasis, has been shown toselectively stimulate Th2 cytokine secretion from PBMC (de Jong R;Bezemer A C; Zomerdijk T P; van de Pouw-Kraan T; Ottenhoff T H;Nibbering P H, Selective stimulation of T helper 2 cytokine responses bythe anti-psoriasis agent monomethylfumarate, Eur J Immunol, 26:2067-2074(1996)). Therefore, IL-4 would be expected to reverse the Thpolarization and be of clinical benefit in psoriasis.

[0011] Certain infectious diseases are associated with polarized Th cellresponses to the infectious agent. Th2 responses have in some cases beenassociated with resistance to the infectious agent. An example isBorrelia burgdorfei, the infectious agent for Lyme disease. Humansinfected with B. burgdorferi exhibit a predominantly Th1-like cytokineprofile (Oksi J; Savolainen J; Pene J; Bousquet J; Laippala P; ViljanenM K, Decreased interleukin-4 and increased gamma interferon productionby peripheral blood mononuclear cells of patients with Lyme borreliosis,Infect. Immun., 64:3620-3623 (1996)). In a mouse model of B.burgdoreri-induced arthritis, resistance to disease is associated withIL-4 production while susceptibility is associated with INF-γ production(Matyniak J E; Reiner S L, T helper phenotype and genetic susceptibilityin experimental Lyme disease, J Exp Med, 181(3):1251-1254 (1995);Keane-Myers A; Nickell S P, Role of IL-4 and IFN-gamma in modulation ofimmunity to Borrelia burgdorferi in mice, J Immunol, 155:2020-2028(1995)). Treatment of B. burgdorferi-infected mice with IL-4 augmentsresistance to the infection (Keane-Myers A; Maliszewski C R; Finkelman FD; Nickell S P, Recombinant IL-4 treatment augments resistance toBorrelia burgdorferi infections in both normal susceptible andantibody-deficient susceptible mice, J Immunol., 156:2488-2494(1996)).

[0012] IL-4 has been reported to have a direct effect on inhibiting thegrowth of lymphomas and leukemias (Akashi, K, The role of interleukin-4in the negative regulation of leukemia cell growth, Leuk Lymphoma,9:205-9 (1993)). For example, IL-4 has been reported to induce apoptosisin cells from patient, with acute lymphoblastic leukemia (Manabe, A, etal., Interleukin-4 induces programmed cell death (apoptosis) in cases ofhigh-risk acute lymphoblastic leukemia, Blood, 83:1731-7 (1994)), andinhibits the growth of cells from patients with non-Hodgkin's B celllymphoma (Defrance, T, et al., Antiproliferative effects ofinterleukin-4 on freshly isolated non-Hodgkin malignant B-lymphomacells, Blood, 79:990-6 (1992)).

[0013] IL-4 has also been reported to exhibit activities which suggeststhat it would be of clinical benefit in osteoarthritis. Osteoarthritisis a disease in which the degradation of cartilage is the primarypathology (Sack, K E, Osteoarthritis, A continuing challenge, West JMed, 163:579-86 (1995); Oddis, C V, New perspectives on osteoarthritis,Am J Med, 100:10S-15S (1996)). IL-4 inhibits TNF-α and IL-1 betaproduction by monocytes and synoviocytes from osteoarthritic patients(Bendrups, A, Hilton, A, Meager, A and Hamilton, J A, Reduction of tumornecrosis factor alpha and interleukin-1 beta levels in human synovialtissue by interleukin-4 and glucocorticoid, Rheumatol Int, 12:217-20(1993); Seitz, M, et al., Production of interleukin-1 receptorantagonist, inflammatory chemotactic proteins, and prostaglandin E byrheumatoid and osteoarthritic synoviocytes—regulation by IFN-gamma andIL-4, J Immunol, 152:2060-5 (1994)). Additionally, IL-4 has beenreported to directly block the degradation of cartilage in ex vivocartilage explants (Yeh, L A, Augustine, A J, Lee, P, Riviere, L R andSheldon, A, Interleukin-4, an inhibitor of cartilage breakdown in bovinearticular cartilage explants, J Rheumatol, 22:1740-6 (1995)). Theseactivities suggest that IL-4 would be of clinical benefit inosteoarthritis.

[0014] However, the clinical use of IL-4 has been limited due to itsacute toxicity, which is manifested as a vascular leak syndrome(Margolin, K, et al., Phase II Studies of Human RecombinantInterleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J.Immunotherapy, 15:147-153 (1994)). There is no art in the literaturewhich describes the mechanism of the acute toxic effect of IL-4, northat describes analogs or mutants of IL-4 that retain immunoregulatoryactivities but have reduced acute toxicity.

[0015] IL-4 mutant proteins (“muteins”) are known. The IL-4 muteinIL-4/Y124D is a T cell antagonist (Kruse N, Tony H P, Sebald W,Conversion of human interleukin-4 into a high affinity antagonist by asingle amino acid replacement, Embo J, 11:3237-44 (1992)).

[0016] Therapeutic uses of IL-4 found in patents or patent applicationsinclude the following: the use of IL-4 for potentiation of anticancereffects of chemotherapeutic agents, particularly Hodgkin's Disease andnon-Hodgkins Lymphoma (see WO 9607422); the use of antigenic fragmentsof IL-4 to generate antibodies to treat IL-4 related diseases bysuppressing or imitating the binding activity of IL-4 (see WO 9524481),and to detect, measure and immunopurify IL-4 (see WO 9317106); forinducing the differentiation of precursor B cells to Immunoglobulinsecreting cells, the mature B cells being useful for restoring immunefunction in immune-compromised patients (see WO 9404658); when used incombination with IL-10, as a therapy for treatment of leukemia,lymphoma, inflammatory bowel disease and delayed type hypersensitivity(e.g. ulcerative colitis and Crohn's Disease)(see WO 9404180); treatmentof HIV infection by administering IL-4 to inhibit viral replication inmonocytes and macrophages, and to increase their cytotoxicity towardssome tumor cells (see WO 9404179); for stimulation of skin fibroblastproliferation for treating wounds in diabetic and immuno-compromisedpatients (see WO 9211861); for enhancing the primary immune responsewhen administering bacterial, toxoid, and viral vaccines, especiallytetanus toxoid vaccine (see WO 9211030); for inhibition of IL-2 inducedproliferation of B cell malignancies, especially chronic lymphocyticleukemia, non-Hodgkin's malignant lymphoma (see WO 9210201); use of IL-4to treat melanomas, renal and basal cell carcinomas (see WO 9204044).

[0017] The patent literature discloses IL-4 proteins and some muteins,but none directed to an IL-4 therapy with reduced side effects. Lee etal. U.S. Pat. No. 5,017,691 (“the '691 patent”) is directed to mammalianproteins and muteins of human IL-4 which disclose both B-cell growthfactor activity and T cell growth factor activity. It discloses nucleicacids coding for polypeptides exhibiting IL-4 activity, as well as thepolypeptides themselves and methods for their production. Muteins to thewild-type IL-4 at amino acid positions are disclosed that retain theirability to stimulate both B- and T cell proliferation in vitro. However,nothing in Lee suggests any T cell selective IL-4 muteins, anticipatedactivation of EC's or the endothelial cell leakiness which accompaniesadministration of IL-4. Thus, IL-4 itself is not enabling as atherapeutic modality because of the dose-limiting toxicity.

[0018] U.S. Pat. No. 5,013,824 describes hIL-4 peptide derivativescomprising from 6 to 40 amino acids of the native hIL-4. Also disclosedare immunogens comprising conjugates of the peptides and carriers.Carriers include erythrocytes, bacteriophages, proteins, syntheticparticles or any substance capable of eliciting antibody productionagainst the conjugated peptide. No muteins of IL-4 are disclosed.

[0019] WO96/04306-A2 discloses single-muteins that are antagonists andpartial agonists of hIL-2 and hIL-13. No data regarding IL-4 isdisclosed. WO95/27052 discloses splice mutants of IL-2 and IL-4containing exons 1, 2 and 4.

[0020] There exists a need for an improved IL-4 molecule which hasreduced toxicity and is more generally tolerated.

SUMMARY OF THE INVENTION

[0021] The invention is directed to human IL-4 muteins numbered inaccordance with wild-type IL-4 having T cell activating activity, buthaving reduced endothelial cell activating activity. In particular,human IL-4 muteins wherein the surface-exposed residues of the D helixof the wild-type IL-4 are mutated whereby the resulting mutein causes Tcell proliferation, and causes reduced IL-6 secretion from HUVECs,relative to wild-type IL-4. This invention realizes a less toxic IL-4mutein that allows greater therapeutic use of this interleukin.

[0022] Further, the invention is directed to IL-4 muteins having single,double and triple mutations represented by the designators R121A, R121D,R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W; Y124A,Y124Q, Y124R, Y124S, Y124T; Y124A/S125A, T13D/R121E; andR121T/E122F/Y124Q, when numbered in accordance with wild type IL-4(His=1). The invention also includes polynucleotides coding for themuteins of the invention, vectors containing the polynucleotides,transformed host cells, pharmaceutical compositions comprising themuteins, and therapeutic methods of treatment.

[0023] The invention is also directed to a vector comprising thepolynucleotide encoding a mutein of this invention, the vector directingthe expression of a human IL-4 mutein having T cell activating activitybut having reduced endothelial cell activating activity, the vectorbeing capable of enabling transfection of a target organism andsubsequent in vivo expression of said human IL-4 mutein coded for bysaid polynucleotide.

[0024] The invention is also directed to a method of selecting a humanIL-4 mutein numbered in accordance with wild-type IL-4 having T cellactivating activity but having reduced endothelial cell activatingactivity, comprising mutating the surface-exposed residues of the Dhelix of the wild-type IL-4 whereby the resulting mutein causes T cellproliferation, and causes reduced IL-6 secretion from HUVECs, relativeto wild-type.

[0025] The invention is also directed to a method of treating a patientafflicted with an IL-4 treatable condition by administering atherapeutically effective amount of a human IL-4 mutein numbered inaccordance with wild-type IL-4 having T cell activating activity buthaving reduced endothelial cell activating activity. This method isapplicable wherein the IL-4 treatable condition is an autoimmunedisorder, cancer, infectious disease, cartilage disorder, and psoriaticdisorders.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is an amino acid sequence (SEQ ID NO:1) of mature wild-typehuman IL-4 used in this study. Helices are underlined and labeledsequentially A, B, C, and D. Positions that, when mutated yieldedcell-selective IL-4 agonists, are indicated in bold type.

[0027]FIG. 2 is a graphical presentation of the T cell selective agonistconcept.

[0028]FIG. 3 is a composite dose response curve for selective agonistmuteins in the HUVEC IL-6 secretion assay. Panel A: O, wild-type IL-4;, R121E; ∇, R121P; ▾, R121T/E122F/Y124Q. Panel B: O, wild-type IL-4; ,Y124Q; ∇, Y124R; ▾, Y124A/S125A.

[0029]FIG. 4 are individual dose response curves of selective agonistmuteins in the HUVEC IL-6 secretion assay. Panel A: O, wild-type IL-4;, R121E. Panel B: O, wild-type IL-4; , R121P. Panel C: O, wild-typeIL-4; , Y124Q. Panel D: O, wild-type IL-4; , Y124R. Panel E: O,wild-type IL-4; , Y124A/S125A. Panel F: O, wild-type IL-4; ,R121T/E122F/Y124Q.

[0030]FIG. 5 is a composite dose-response curve for selective agonistmuteins for the biological response of IL-4 muteins in 1° T cellproliferation assays. Panel A: O, wild-type IL-4; , R121E; ∇, R121P; ▾,R121T/E122F/Y124Q. Panel B: O, wild-type IL-4; , Y124Q; ∇, Y124R; ▾,Y124A/S125A.

[0031]FIG. 6 are individual dose response curves of the 1° T cellproliferation assay. Panel A: O, wild-type IL-4; , R121E. Panel B: O,wild-type IL-4; , R121P. Panel C: O, wild-type IL-4; , Y124Q. Panel D:O, wild-type IL-4; , Y124R. Panel E: O, wild-type IL-4; , Y124A/S125A.Panel F: O, wild-type IL-4; , R121T/E122F/Y124Q.

[0032]FIG. 7 are individual dose response curves showing the antagonismof IL-4-induced IL-6 secretion on HUVEC by the T cell-selective agonistIL-4 muteins R121E () and Y124Q (∇). The dose response of the IL-4antagonist R121D/Y124D (O) is included as a control.

[0033]FIGS. 8A and 8B are individual dose response curves: Panel A showsthe biological response of the R121D mutein in a 1° T cell proliferationassay (O=IL-4, =R121D); Panel B shows the inability of R121D to induceIL-6 secretion on HUVEC (O=IL-4, =R121D).

[0034]FIG. 9A are the individual dose response curves for IL-4 (O) andthe T cell-selective agonist muteins R121E (Δ) and T13D/R121E (▴) in the1° T cell proliferation assay.

[0035]FIG. 9B are individual dose response curves showing the antagonismof IL-4-induced IL-6 secretion on HUVEC by the T cell-selective IL-4agonist muteins R121E (Δ) and T13D/R121E (▴).

[0036]FIG. 9C is an individual dose response curve showing % maximumcytokine release versus concentration. In the HUVEC assay shown,T13D/R121E (▴) exhibited nominal activity comparable to that seen withR121E (Δ). In this assay, measurement of monocyte chemoattractantprotein 1 (MCP-1) in the media by ELISA was used instead of IL-6 tomeasure activity.

[0037]FIGS. 10A through 10D are graphs that show the change in Day 1levels of fibrinogen and APTT as a function of exposure to test article.

[0038] FIGS. 11A-B are graphs of percentages of CD23+ and CD4+IL-4R+over CD4+ lymphocytes in the peripheral blood during Phase II of thesafety pharmacology study. Each point plotted corresponds to theaverage±s.d. of observations from 2 animals.

[0039] FIGS. 12A-12C are charts showing FACS analysis for CD4+, showinglymphocytes in the circulation, Phase I (FIG. 12A), Phase II (FIG. 12B).FIG. 12C is a histogram of the effects of vehicle, 10 and 30 μg/kgwild-type IL-4, and 10, 30, 100 and 300 μg/kg IL-4[T13D/R121E].

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] A. Background

[0041] IL-4 has been shown to mediate a variety of cellular responses invitro, including various effects on B cells, T cells, and monocytes, aswell as endothelial cells (Maher D W, Davis I, Boyd A W, Morstyn G:Human interleukin-4: an immunomodulator with potential therapeuticapplications. Prog Growth Factor Res 3:43-56, 1991; Powrie F, Coffman RL: Cytokine regulation of T cell function: potential for therapeuticintervention. Immunol Today 14:270-4, 1993). In particular, upregulationof vascular cell adhesion molecule-1 (VCAM-1; (Swerlick R A, Lee K H, LiL J, Sepp N T, Caughman S W, Lawley T J: Regulation of vascular celladhesion molecule 1 on human dermal microvascular endothelial cells. JImmunol 149:698-705, 1992)) and induction of IL-6 (Colotta F, Sironi M,Borre A, Luini W, Maddalena F, Mantovani A: Interleukin 4 amplifiesmonocyte chemotactic protein and interleukin 6 production by endothelialcells. Cytokine 4:24-8, 1992) and monocyte chemoattractant protein-1(MCP-1; Colotta F, Sironi M, Borre A, Luini W, Maddalena F, Mantovani A:Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6production by endothelial cells. Cytokine 4:24-8, 1992; Rollins B J,Pober J S: Interleukin-4 induces the synthesis and secretion ofMCP-1I/JE by human endothelial cells. Am J Pathol 138:1315-9, 1991)) aredirect effects of IL-4 on cultured endothelial cells; the upregulationof VCAM-1 is correlated with the increased adhesion of lymphocytes bothin vitro (Carlos T M, Schwartz B R, Kovach N L, Yee E, Rosa M, Osborn L,Chi-Rosso G, Newman B, Lobb R, Rosso M, et al.: Vascular cell adhesionmolecule-1 mediates lymphocyte adherence to cytokine-activated culturedhuman endothelial cells. Blood 76:965-70, 1990; Thornhill M H, WellicomeS M, Mahiouz D L, Lanchbury J S, Kyan-Aung U, Haskard D O: Tumornecrosis factor combines with IL-4 or IFN-gamma to selectively enhanceendothelial cell adhesiveness for T cells. The contribution of vascularcell adhesion molecule-1-dependent and -independent binding mechanisms.J Immunol 146:592-8, 1991) and in vivo (Briscoe D M, Cotran R S, Pober JS: Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on theexpression of vascular cell adhesion molecule-1 in vivo. Correlationwith CD3+ T cell infiltration. J Immunol 149:2954-60, 1992).

[0042] The IL-4 mutein IL-4/Y124D (substitution of Aspartic acid forTyrosine at position 124) is a T cell antagonist (Kruse N, Tony H P,Sebald W: Conversion of human interleukin-4 into a high affinityantagonist by a single amino acid replacement. Embo J 11:3237-44, 1992).In vivo experiments performed by the inventors have demonstrated thatIL-4/Y124D exhibits acute toxicity similar to that of wild-type IL-4 inmonkeys, a previously undescribed observation. The cellular eventsassociated with both wild-type IL-4- and IL-4/Y124D-mediated toxicityinclude upregulation of VCAM-1, upregulation of MCP-1 in serum,increases in circulating monocytes together with a concomitant decreasein circulating lymphocytes, and an increase in hematocrit. Similarcellular trafficking has been observed in clinical trials using IL-4 inhumans (Wong H L, Lotze M T, Wahl L M, Wahl S M: Administration ofrecombinant IL-4 to humans regulates gene expression, phenotype, andfunction in circulating monocytes. J Immunol 148:2118-25, 1992). Due toits properties as an antagonist of T cells, these results suggest thatthe toxicities demonstrated by IL-4/Y124D are due to agonist activitieson and are mediated through cells other than T cells. The observed invivo toxicities using IL-4/Y124D and the known effects of IL-4 onendothelial cells are consistent with the mechanism that in vivo IL-4toxicity is mediated through direct effects of IL-4 on the vascularendothelium.

[0043] Through these (and related) investigations, the inventors havediscovered that a new IL-4 receptor may exist on endothelial cells(“EC”). This possibility led to efforts to synthesize IL-4 muteins thatwould selectively activate T cells, but not EC. While T cells express anIL-4 receptor composed of IL-4Rα and IL-2Rγ subunits, the inventors havediscovered that human umbilical vein endothelial cells (HUVEC), expressIL-4Rα but not IL-2Rγ. Crosslinking studies have shown that two receptorchains are expressed at the cell surface of HUVEC: the molecular weightof one is consistent with IL-4Rα, and a second, lower molecular weightchain. These results suggest that a novel IL-4 receptor componentsimilar in function to IL-2Rγ, but differing in sequence, is expressedon HUVECs. The differences in the specific molecular structures betweenthese two receptors were thus exploited to generate an IL-4 variant thatis selective for one receptor over the other (e.g., a T cell selectiveagonist).

[0044]FIG. 2 demonstrates graphically the selective agonist concept. Itshows the T cell IL-4 receptor comprising the IL-4Rα/IL-2Rγ subunit, andan Endothelial cell IL-4 receptor comprising the IL-4Rα/γ-like receptorsubunit. Although depicted here together for purposes of illustrationonly, the two receptors for IL-4 are expressed on different cell types.The T cell receptor is composed of IL-4Rα and IL-2Rγ; IL-4 bindinginduces receptor heterodimer formation that results in cellularsignalling. IL-4 induced receptor heterodimer formation occurs in asimilar manner on EC's, except that the receptor for IL-4 is composed ofIL-4Rα and a γ-like receptor component. The γ-like receptor component isdifferent from IL-2Rγ. T cell-selective IL-4 agonists are those variantsof IL-4 that retain their ability to interact with the T cell receptorIL-4Rα/IL-2Rγ, but are unable to induce heterodimerization, and thussignalling, of the non-T cell receptor IL-4Rα/γ-like subunit. Such Tcell-selective IL-4 agonists retain their ability to interact withIL-4Rα; it is their ability to discriminate between IL-2Rγ and theγ-like subunit that gives them their cell-selective activationproperties.

[0045] The two components of the T cell receptor, IL4-Rα and IL-2Rγ,contact different regions of the IL-4 molecule, and therefor theinventors have focussed on a small region of IL-4 to modify.Hypothesizing that the novel receptor subunit would contact the sameregion of IL4 as does IL-2Rγ, the inventors made a number ofsubstitutions in the D-Helix, particularly residues 121, 124 and 125.

[0046] The D-helix has been implicated in interactions with both IL-2Rγand with the putative novel receptor on HUVEC (specifically, the IL-4mutein R121D/Y124D is a HUVEC antagonist). Muteins containingmodifications to the D-helix of IL-4 (residues 110 to 126; His=1) werescreened for their ability to stimulate either T cell proliferation orhuman umbilical vein endothelial cell (HUVEC) secretion of IL-6. Muteinsthat induced a differential response on T cells relative to HUVEC werefurther characterized through further mutagenesis.

[0047] An initial scan of the D helix was undertaken to determine thepotential areas of interaction. Additionally, alanine scanningsubstitutions of the AB loop were also generated, as this region issuggested to be involved in the interaction of the cytokine ligand andthe D-helix interacting receptor subunit. In particular, surface-exposedresidues Glu-110, Asn-111, Glu-114, Arg-115, Lys-117, Thr-118, Arg-121,Glu-122, Tyr-124, Ser-125, and Lys-126 were targeted for investigationand are preferred targets for mutation analysis. Sites 118-126 are morepreferred, and sites 121-125 are most preferred. Comparisons betweenIL-2, IL-4, IL-7 and IL-15 in this region also identify differencesbetween IL-4 and IL-2, IL-7 and IL-15, possibly suggesting specificresidues responsible for the HUVEC receptor interaction. Specificsubstitutions derived from an alignment between IL-2 and IL-4 wereintroduced into IL-4. These included: Arg-115 to Phe; Lys-117 to Asn;Glu-122 to Phe; Lys-126 to Ile; and three simultaneous changes Arg-121to Thr, Glu-122 to Phe, and Tyr-124 to Gln.

[0048] Mutations were introduced using site-directed mutagenesis onwild-type human IL-4 cDNA. Correct clones were subcloned to anexpression vector suitable for expression in a heterologous system(e.g., E. coli, baculovirus, or CHO cells). Purified proteins weretested in T cell proliferation and HUVEC cytokine secretion assays(IL-6). Different responses generated by individual muteins betweenthese assays, either in EC₅₀ or maximal response (plateau) indicatemutations that effect these activities. Specifically, muteins thatstimulate a relatively stronger response in the T cell assay (vs.wild-type IL-4) as compared to the response on HUVEC (vs. wild-typeIL-4) will suggest positions that are more important to the interactionof IL-4 with IL-2Rγ than the interaction of IL-4 with the novel HUVECIL-4 receptor. Further analysis and mutagenesis (e.g. combinatorialchanges, substitution with all amino acids) of the identified positionswill produce an IL-4 mutein with selective agonist properties for the Tcell IL-4 receptor. This protein will also be a selective antagonist forIL-4-induced HUVEC responses.

[0049] B. Definitions

[0050] Described herein are novel muteins and a mechanism for derivingnovel IL-4 muteins with selective agonist properties on T cells andreduced toxicity. A similar strategy may be used to identify a Tcell-selective antagonist.

[0051] As used herein, “wild type IL-4” means IL-4, whether native orrecombinant, having the 129 normally occurring amino acid sequence ofnative human IL-4, as shown, e.g., in FIG. 1.

[0052] As used herein, “IL-4 mutein” means a polypeptide whereinspecific substitutions to the human mature interleukin-4 protein havebeen made. Specifically disclosed herein, the arginine residue (R) atposition 121 (“Arg-121”), when numbered in accordance with wild typeIL-4, is substituted with alanine (A), aspartate (D), glutamate (E),phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine(N) proline (P), threonine (T) or tryptophan (W); or the glutamate (E)residue at position 122 is substituted with phenylalanine (F); or thetyrosine residue at position 124 is substituted with alanine (A),glutamine (Q), arginine (R) serine (S) or threonine (T); or the serine(S) residue at position 125 is substituted with alanine (A). Our mostpreferred IL-4 muteins have an amino acid sequence identical to wildtype IL-4 at the other, non-substituted residues. However, the IL-4muteins of this invention may also be characterized by amino acidinsertions, deletions, substitutions and modifications at one or moresites in or at the other residues of the native IL-4 polypeptide chain.In accordance with this invention any such insertions, deletions,substitutions and modifications result in an IL-4 mutein that retains aT cell-selective activity while having reduced ability to activateendothelial cells.

[0053] We prefer conservative modifications and substitutions at otherpositions of IL-4 (i.e., those that have a minimal effect on thesecondary or tertiary structure of the mutein). Such conservativesubstitutions include those described by Dayhoff in The Atlas of ProteinSequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785(1989). For example, amino acids belonging to one of the followinggroups represent conservative changes:

[0054] ala, pro, gly, gln, asn, ser, thr;

[0055] cys, ser, tyr, thr;

[0056] val, ile, leu, met, ala, phe;

[0057] lys, arg, his;

[0058] phe, tyr, trp, his; and

[0059] asp, glu.

[0060] We also prefer modifications or substitutions that do notintroduce sites for additional intermolecular crosslinking or incorrectdisulfide bond formation. For example, IL-4 is known to have six cysresidues, at wild-type positions 3, 24, 46, 65, 99 and 127.

[0061] By “numbered in accordance with wild type IL-4” we meanidentifying a chosen amino acid with reference to the position at whichthat amino acid normally occurs in wild type IL-4. Where insertions ordeletions are made to the IL-4 mutein, one of skill in the art willappreciate that the ser (S) normally occurring at position 125, whennumbered in accordance with wild type IL-4, may be shifted in positionin the mutein. However, the location of the shifted ser (S) can bereadily determined by inspection and correlation of the flanking aminoacids with those flanking ser in wild type IL-4.

[0062] The IL-4 muteins of the present invention can be produced by anysuitable method known in the art. Such methods include constructing aDNA sequence encoding the IL-4 muteins of this invention and expressingthose sequences in a suitably transformed host. This method will producerecombinant muteins of this invention. However, the muteins of thisinvention may also be produced, albeit less preferably, by chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

[0063] In one embodiment of a recombinant method for producing a muteinof this invention, a DNA sequence is constructed by isolating orsynthesizing a DNA sequence encoding the wild type IL-4 and thenchanging the codon for arg121 to a codon for alanine (A), aspartate (D),glutamate (E), phenylalanine (F), histidine (H), isoleucine (I), lysine(K), asparagine (N) proline (P), threonine (T) or tryptophan (W) bysite-specific mutagenesis. This technique is well known. See, e.g., Market al., “Site-specific Mutagenesis Of The Human Fibroblast InterferonGene”, Proc. Natl. Acad. Sci. USA 81, pp. 5662-66 (1984); U.S. Pat. No.4,588,585, incorporated herein by reference.

[0064] Another method of constructing a DNA sequence encoding the IL-4muteins of this invention would be chemical synthesis. For example, agene which encodes the desired IL-4 mutein may be synthesized bychemical means using an oligonucleotide synthesizer. Sucholigonucleotides are designed based on the amino acid sequence of thedesired IL-4 mutein, and preferably selecting those codons that arefavored in the host cell in which the recombinant mutein will beproduced. In this regard, it is well recognized that the genetic code isdegenerate—that an amino acid may be coded for by more than one codon.For example, phe (F) is coded for by two codons, TTC or TTT, tyr (Y) iscoded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W)is coded for by a single codon, TGG. Accordingly, it will be appreciatedthat for a given DNA sequence encoding a particular IL-4 mutein, therewill be many DNA degenerate sequences that will code for that IL-4mutein. For example, it will be appreciated that in addition to thepreferred DNA sequence for mutein R121E shown in SEQ ID NO:3, there willbe many degenerate DNA sequences that code for the IL-4 mutein shown.These degenerate DNA sequences are considered within the scope of thisinvention. Therefore, “degenerate variants thereof” in the context ofthis invention means all DNA sequences that code for a particularmutein.

[0065] The DNA sequence encoding the IL-4 mutein of this invention,whether prepared by site directed mutagenesis, synthesis or othermethods, may or may not also include DNA sequences that encode a signalsequence. Such signal sequence, if present, should be one recognized bythe cell chosen for expression of the IL-4 mutein. It may beprokaryotic, eukaryotic or a combination of the two. It may also be thesignal sequence of native IL-4. The inclusion of a signal sequencedepends on whether it is desired to secrete the IL-4 mutein from therecombinant cells in which it is made. If the chosen cells areprokaryotic, it generally is preferred that the DNA sequence not encodea signal sequence. If the chosen cells are eukaryotic, it generally ispreferred that a signal sequence be encoded and most preferably that thewild-type IL-4 signal sequence be used.

[0066] Standard methods may be applied to synthesize a gene encoding anIL-4 mutein according to this invention. For example, the complete aminoacid sequence may be used to construct a back-translated gene. A DNAoligomer containing a nucleotide sequence coding for IL-4 mutein may besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

[0067] Once assembled (by synthesis, site-directed mutagenesis oranother method), the DNA sequences encoding an IL-4 mutein of thisinvention will be inserted into an expression vector and operativelylinked to an expression control sequence appropriate for expression ofthe IL-4 mutein in the desired transformed host. Proper assembly may beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

[0068] The choice of expression control sequence and expression vectorwill depend upon the choice of host. A wide variety of expressionhost/vector combinations may be employed. Useful expression vectors foreukaryotic hosts, include, for example, vectors comprising expressioncontrol sequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including colE1, pCR1, pER32z, pMB9 and their derivatives, wider host range plasmids,such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda,e.g., NM989, and other DNA phages, such as M13 and filamentous singlestranded DNA phages. Useful expression vectors for yeast cells includethe 2μ plasmid and derivatives thereof. Useful vectors for insect cellsinclude pVL 941. We prefer pFastBac™ 1 (GibcoBRL, Gaithersburg, Md.).Cate et al., “Isolation Of The Bovine And Human Genes For MullerianInhibiting Substance And Expression Of The Human Gene In Animal Cells”,Cell, 45, pp. 685-98 (1986).

[0069] In addition, any of a wide variety of expression controlsequences may be used in these vectors. Such useful expression controlsequences include the expression control sequences associated withstructural genes of the foregoing expression vectors. Examples of usefulexpression control sequences include, for example, the early and latepromoters of SV40 or adenovirus, the lac system, the trp system, the TACor TRC system, the major operator and promoter regions of phage lambda,for example PL, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., PhoA, the promoters of the yeast α-matingsystem, the polyhedron promotor of Baculovirus, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells or their viruses, and various combinations thereof.

[0070] Any suitable host may be used to produce the IL-4 muteins of thisinvention, including bacteria, fungi (including yeasts), plant, insect,mammal, or other appropriate animal cells or cell lines, as well astransgenic animals or plants. More particularly, these hosts may includewell known eukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such asSpodoptera frugiperda (Sf9), animal cells such as Chinese hamster ovary(CHO) and mouse cells such as NS/O, African green monkey cells such asCOS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells, as well asplant cells in tissue culture. For animal cell expression, we prefer CHOcells and COS 7 cells in cultures and particularly the CHO cell line CHO(D HFR-).

[0071] It should of course be understood that not all vectors andexpression control sequences will function equally well to express theDNA sequences described herein. Neither will all hosts function equallywell with the same expression system. However, one of skill in the artmay make a selection among these vectors, expression control sequencesand hosts without undue experimentation. For example, in selecting avector, the host must be considered because the vector must replicate init. The vector's copy number, the ability to control that copy number,and the expression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. For example, preferredvectors for use in this invention include those that allow the DNAencoding the IL-4 muteins to be amplified in copy number. Suchamplifiable vectors are well known in the art. They include, forexample, vectors able to be amplified by DHFR amplification (see, e.g.,Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, “Construction Of AModular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals UtilizedFor Efficient Expression”, Mol. Cell. Biol., 2, pp. 1304-19 (1982)) orglutamine synthetase (“GS”) amplification (see, e.g., U.S. Pat. No.5,122,464 and European published application 338,841).

[0072] In selecting an expression control sequence, a variety of factorsshould also be considered. These include, for example, the relativestrength of the sequence, its controllability, and its compatibilitywith the actual DNA sequence encoding the IL-4 mutein of this invention,particularly as regards potential secondary structures. Hosts should beselected by consideration of their compatibility with the chosen vector,the toxicity of the product coded for by the DNA sequences of thisinvention, their secretion characteristics, their ability to fold thepolypeptides correctly, their fermentation or culture requirements, andthe ease of purification of the products coded for by the DNA sequences.

[0073] Within these parameters, one of skill in the art may selectvarious vector/expression control sequence/host combinations that willexpress the desired DNA sequences on fermentation or in large scaleanimal culture, for example, using CHO cells or COS 7 cells.

[0074] The IL-4 muteins obtained according to the present invention maybe glycosylated or unglycosylated depending on the host organism used toproduce the mutein. If bacteria are chosen as the host then the IL-4mutein produced will be unglycosylated. Eukaryotic cells, on the otherhand, will glycosylate the IL-4 muteins, although perhaps not in thesame way as native IL-4 is glycosylated.

[0075] The IL-4 mutein produced by the transformed host can be purifiedaccording to any suitable method. Various methods are known forpurifying IL-4. See, e.g., U.S. Pat. Nos. 5,013,824; 5,017,691; andWO9604306-A2. We prefer immunoaffinity purification. See, e.g., Okamuraet al., “Human Fibroblastoid Interferon: Immunosorbent ColumnChromatography And N-Terminal Amino Acid Sequence”, Biochem., 19, pp.3831-35 (1980).

[0076] The biological activity of the IL-4 muteins of this invention canbe assayed by any suitable method known in the art. Such assays includeantibody neutralization of antiviral activity, induction of proteinkinase, oligoadenylate 2,5-A synthetase or phosphodiesterase activities,as described in EP-B1-41313. Such assays also include immunomodulatoryassays (see, e.g., U.S. Pat. No. 4,753,795), growth inhibition assays, Tcell proliferation, induction of IL-6 (MCP-1 or VCAM-1) on EC andmeasurement of binding to cells that express interleukin-4 receptors.See also Spits H, Yssel H, Takebe Y; et al., Recombinant Interleukin-4Promotes the Growth of Human T Cells, J. IMMUNOL 139:1142-47 (1987).

[0077] The IL-4 mutein of this invention will be administered at a doseapproximately paralleling that or greater than employed in therapy withwild type native or recombinant IL-4. An effective amount of the IL-4mutein is preferably administered. An “effective amount” means an amountcapable of preventing or lessening the severity or spread of thecondition or indication being treated. It will be apparent to those ofskill in the art that the effective amount of IL-4 mutein will depend,inter alia, upon the disease, the dose, the administration schedule ofthe IL-4 mutein, whether the IL-4 mutein is administered alone or inconjunction with other therapeutic agents, the serum half-life of thecomposition, and the general health of the patient.

[0078] The IL-4 mutein is preferably administered in a compositionincluding a pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” means a carrier that does not cause any untowardeffect in patients to whom it is administered. Such pharmaceuticallyacceptable carriers are well known in the art. We prefer 2% HSA/PBS atpH7.0.

[0079] The IL-4 muteins of the present invention can be formulated intopharmaceutical compositions by well known methods. See, e.g.,Remington's Pharmaceutical Science by E. W. Martin, hereby incorporatedby reference, describes suitable formulations. The pharmaceuticalcomposition of the IL-4 mutein may be formulated in a variety of forms,including liquid, gel, lyophilized, or any other suitable form. Thepreferred form will depend upon the particular indication being treatedand will be apparent to one of skill in the art.

[0080] The IL-4 mutein pharmaceutical composition may be administeredorally, by aerosol, intravenously, intramuscularly, intraperitoneally,intradermally or subcutaneously or in any other acceptable manner. Thepreferred mode of administration will depend upon the particularindication being treated and Will be apparent to one of skill in theart. The pharmaceutical composition of the IL-4 mutein may beadministered in conjunction with other therapeutic agents. These agentsmay be incorporated as part of the same pharmaceutical composition ormay be administered separately from the IL-4 mutein, either concurrentlyor in accordance with any other acceptable treatment schedule. Inaddition, the IL-4 mutein pharmaceutical composition may be used as anadjunct to other therapies.

[0081] Accordingly, this invention provides compositions and methods fortreating immune disorders, cancers or tumors, abnormal cell growth, orfor immunomodulation in any suitable animal, preferably a mammal, mostpreferably human. As previously noted in the Background section, IL-4has many effects. Some of these are stimulation of T cell proliferation,T-helper cell differentiation, induction of human B-cell activation andproliferation, and lymphokine-directed immunoglobulin class switching.Effects on the lymphoid system include increasing the expression of MHCclass II antigen (Noelle, R., et al., Increased Expression of IaAntigens on resting B cells: a New Role for B Cell Growth Factor, PNASUSA, 81:6149-53 (1984)), and CD 23 on B cells (Kikutani, H., et al.,Molecular Structure of Human Lymphocyte Receptor for Immunoglobulin,Cell 47:657-61 (1986)). T-helper cell type 1 (Th1) and type 2 (Th2) areinvolved in the immune response. Stimulated Th2 cells secrete IL-4 andblock Th1 progression. Thus, any Th1-implicated disease is amenable totreatment by IL-4 or analogs thereof.

[0082] Also contemplated is use of the DNA sequences encoding the IL-4muteins of this invention in gene therapy applications. Gene therapyapplications contemplated include treatment of those diseases in whichIL-4 is expected to provide an effective therapy due to itsimmunomodulatory activity, e.g., Multiple Sclerosis (MS),Insulin-dependent Diabetes Mellitus (IDDM), Rheumatoid Arthritis (RA),Systemic Lupus Erythematosus (SLE), uveitis, orchitis, primary biliarycirrhosis, malaria, leprosy, Lyme Disease, contact dermatitis,psoriasis, B cell lymphoma, acute lymphoblastic leukemia, non-Hodgkinslymphoma, cancer, osteoarthritis and diseases that are otherwiseresponsive to IL-4 or infectious agents sensitive to IL-4-mediatedimmune response.

[0083] Local delivery of IL-4 muteins using gene therapy may provide thetherapeutic agent to the target area. Both in vitro and in vivo genetherapy methodologies are contemplated. Several methods for transferringpotentially therapeutic genes to defined cell populations are known.See, e.g., Mulligan, “The Basic Science Of Gene Therapy”, Science, 260:926-31 (1993). These methods include:

[0084] 1) Direct gene transfer. See, e.g., Wolff et al., “Direct Genetransfer Into Mouse Muscle In Vivo”, Science, 247:1465-68 (1990);

[0085] 2) Liposome-mediated DNA transfer. See, e.g., Caplen at al.,“Liposome-mediated CFTR Gene Transfer To The Nasal Epithelium OfPatients With Cystic Fibrosis”, Nature Med. 3: 39-46 (1995); Crystal,“The Gene As A Drug”, Nature Med. 1:15-17 (1995); Gao and Huang, “ANovel Cationic Liposome Reagent For Efficient Transfection Of MammalianCells”, Biochem. Biophys. Res. Comm., 179:280-85 (1991);

[0086] 3) Retrovirus-mediated DNA transfer. See, e.g., Kay et al., “InVivo Gene Therapy Of Hemophilia B: Sustained Partial Correction InFactor IX-Deficient Dogs”, Science, 262:117-19 (1993); Anderson, “HumanGene Therapy”, Science, 256:808-13 (1992).

[0087] 4) DNA Virus-mediated DNA transfer. Such DNA viruses includeadenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses(preferably herpes simplex virus based vectors), and parvoviruses(preferably “defective” or non-autonomous parvovirus based vectors, morepreferably adeno-associated virus based vectors, most preferably AAV-2based vectors). See, e.g., Ali et al., “The Use Of DNA Viruses AsVectors For Gene Therapy”, Gene Therapy, 1:367-84 (1994); U.S. Pat. No.4,797,368, incorporated herein by reference, and U.S. Pat. No.5,139,941, incorporated herein by reference.

[0088] The choice of a particular vector system for transferring thegene of interest will depend on a variety of factors. One importantfactor is the nature of the target cell population. Although retroviralvectors have been extensively studied and used in a number of genetherapy applications, these vectors are generally unsuited for infectingnon-dividing cells. In addition, retroviruses have the potential foroncogenicity.

[0089] Adenoviruses have the advantage that they have a broad hostrange, can infect quiescent or terminally differentiated cells, such asneurons or hepatocytes, and appear essentially non-oncogenic. See, e.g.,Ali et al., supra, p. 367. Adenoviruses do not appear to integrate intothe host genome. Because they exist extrachromosomally, the risk ofinsertional mutagenesis is greatly reduced. Ali et al., supra, p. 373.

[0090] Adeno-associated viruses exhibit similar advantages asadenoviral-based vectors. However, AAVs exhibit site-specificintegration on human chromosome 19. Ali et al., supra, p.377.

[0091] In a preferred embodiment, the IL-4 mutein-encoding DNA of thisinvention is used in gene therapy for autoimmune diseases such as MS,IDDM, and RA, infectious diseases such as Lyme Disease and Leprosy,cancers, such as non-Hodgkins lymphoma and ALL, cartiledgenous disorderssuch as osteoarthritis, and psoriatic conditions, such as psoriasis.

[0092] According to this embodiment, gene therapy with DNA encoding theIL-4 muteins of this invention is provided to a patient in need thereof,concurrent with, or immediately after diagnosis.

[0093] This approach takes advantage of the selective activity of theIL-4 muteins of this invention to prevent undesired autoimmunestimulation. The skilled artisan will appreciate that any suitable genetherapy vector containing IL-4 mutein DNA may be used in accordance withthis embodiment. The techniques for constructing such a vector areknown. See, e.g., Ohno et al., supra, p. 784; Chang et al., supra, p.522. Introduction of the IL-4 mutein DNA-containing vector to the targetsite may be accomplished using known techniques, e.g., as described inOhno et al., supra, p. 784.

[0094] In order that this invention may be better understood, thefollowing examples are set forth. These examples are for the purpose ofillustration only, and are not to be construed as limiting the scope ofthe invention in any manner.

EXAMPLES

[0095] Generally.

[0096] The amino acid sequence of mature human IL-4 used in this studyis shown below. Amino acids at which substitutions yielded T cellselective agonists are indicated in bold type: His Lys Cys Asp Ile ThrLeu Gln Glu Ile Ile Lys Thr Leu Asn (SEQ ID NO:1)1               5                   10                  15 Ser Leu ThrGlu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr                20                  25                  30 Asp Ile PheAla Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe                35                  40                  45 Cys Arg AlaAla Thr Val Leu Arg Gln Phe Tyr Ser His His Glu                50                  55                  60 Lys Asp ThrArg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg                65                  70                  75 His Lys GlnLeu Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu                80                  85                  90 Trp Gly LeuAla Gly Leu Asn Ser Cys Pro Val Lys Glu Ala Asn                95                  100                 105 Gln Ser ThrLeu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met                110                 115                 120 Arg Glu LysTyr Ser Lys Cys Ser Ser                 125

[0097] Muteins were expressed in a baculovirus system, purified tohomogeneity, and evaluated in biological assays that reflected differentIL-4 receptor usage. Two assays were used to test for selective agonistactivity, IL-4-induced HUVEC IL-6 secretion assay, and 1° T cellproliferation assay for positive IL-4 activity. Compounds that have theability to induce 1° T cell proliferation, yet have a reduced ability toinduce IL-6 secretion, are T cell selective IL-4 agonists and comewithin the scope of this invention. More specifically, human umbilicalvein endothelial cells (HUVEC) were used to assess activity through thealternate IL-4 receptor (IL-4Rα/γ-like receptor component).

Example 1 Production of muteins

[0098] Muteins were generated by site-directed mutagenesis using primerscontaining codons corresponding to the desired mutation essentially asdescribed by Kunkel T A, Roberts J D, and Zakour R A, “Rapid andefficient site-specific mutagenesis without phenotypic selection”(1987), Methods Enzymol 154: 367-382. Briefly, human IL-4 cDNAcontaining the restriction enzyme sites Bam HI and Xba I was subclonedinto the M13 phage vector M13 mp19 (New England Biolabs, Beverly, Mass.)using the same sites. Wild-type IL-4 cDNA was obtained using PolymeraseChain Reaction (“PCR”) from a cDNA pool generated from MRNA isolatedfrom human peripheral blood lymphocytes induced 24 hours with phorbol12-myristate 13-acetate (10 ng/ml). The PCR primers used were, for the5′ end of the IL-4 open reading frame,

[0099] 5′-CGC GGA TCC ATG GGT CTC ACC TCC-3′ (SEQ ID NO:22);

[0100] and for the 3′ end of the IL-4 open reading frame,

[0101] 5′-CGC TCT AGA CTA GCT CGA ACA CTT TGA AT-3′ (SEQ ID NO:23).

[0102] Restriction enzyme sites BamHI (5′-end) and XbaI (3′-end) wereincorporated into each oligonucleotide and are indicated by italics. ThePCR conditions used were 1 minute at 94° C., 1 minute at 58.7° C., and 1minute at 72° C for 25 cycles. The correct IL-4 cDNA sequence soobtained was confirmed by sequencing using the Sequenase® sequencing kit(Amersham Life Sciences, Arlington Heights, Ill.) as described by themanufacturer. Uracil-containing single strand DNA (U-DNA) was obtainedby transforming the E. coli strain CJ236 (Bio-Rad Laboratories,Hercules, Calif.) with IL-4 cDNA-containing M13 mp19. Site-directedmutagenesis utilized in general primers containing 15 nucleotideshomologous to the template U-DNA 5′ to the codon(s) targetted formutagenesis, nucleotides that incorporate the desired change, and anadditional 10 nucleotides homologous to the template U-DNA 3′ of thelast altered nucleotide. The specific primers used were:

[0103] R121A: CTAAAGACGA TCATGGCTGA GAAATATT (SEQ ID NO:24)

[0104] R121D: GCTAAAGACG ATCATGGACG AGAAATATTC (SEQ ID NO:25)

[0105] R121E: GCTAAAGACG ATCATGGAAG AGAAATATTC (SEQ ID NO:26)

[0106] R121F: CTAAAGACGA TCATGTTTGA GAAATATT (SEQ ID NO:27)

[0107] R121H: CTAAAGACGA TCATGCACGA GAAATATT (SEQ ID NO:28)

[0108] R121I: CTAAAGACGA TCATGATAGA GAAATATT (SEQ ID NO:29)

[0109] R121K: CTAAAGACGA TCATGAAAGA GAAATATT (SEQ ID NO:30)

[0110] R121N: CTAAAGACGA TCATGAACGA GAAATATT (SEQ ID NO:31)

[0111] R121P: GCTAAAGACG ATCATGCCAG AGAAATATTC (SEQ ID NO:32)

[0112] R121T: CTAAAGACGA TCATGACTGA GAAATATT (SEQ ID NO:33)

[0113] R121W: CTAAAGACGA TCATGTGGGA GAAATATT (SEQ ID NO:34)

[0114] Y124A: ATCATGAGAG AGAAAGCATC AAAGTGTT (SEQ ID NO:35)

[0115] Y124Q: ATCATGAGAG AGAAACAATC AAAGTGTT (SEQ ID NO:36)

[0116] Y124R: ATCATGAGAG AGAAACGATC AAAGTGTT (SEQ ID NO:37)

[0117] Y124S: ATCATGAGAG AGAAATCATC AAAGTGTT (SEQ ID NO:38)

[0118] Y124T: ATCATGAGAG AGAAAACATC AAAGTGTT (SEQ ID NO:39)

[0119] Y124A/S125A:CGATCATGAG AGAGAAAGCT GCTAAGTGTT CGA (SEQ ID NO:40)

[0120] T13D: CAGGAGATCA TCAAAGATTT GAACAGCC (SEQ ID NO:41)

[0121] R121T/E122F/Y124Q:GCTAAAGACG ATCATGACCT TCAAACAGTC AAAG (SEQ IDNO:42)

[0122] Regions of mutated nucleotides are underlined. Primers werephosphorylated using T4 polynucleotide kinase (New England Biolabs,Beverly, Mass.) using the manufacturer's protocol. After annealing ofthe primer to the U-DNA template and extension with T7 DNA polymerase(Bio-Rad Laboratories, Hercules, Calif.), cells of the E. coli strainDH5α™ (GibcoBRL, Gaithersburg, Md.) were transformed with 5 μl ofreaction mixture and plated in LB medium containing 0.7% agar. Afterincubation at 37° C., plaques were expanded by picking a single plaqueand transferring to 2 mls of LB media and grown overnight at 37° C.Single strand DNA was isolated using an M13 purification kit (Qiagen,Inc., Chatsworth, Calif.) per manufacturer's protocol, and clonescontaining the desired mutation were identified by sequencing the singlestranded DNA using the Sequenase® sequencing kit (Amersham LifeSciences, Arlington Heights, Ill.) per manufacturer's protocol. IL-4mutein cDNA from Replicative Form DNA corresponding to plaquescontaining the correct mutated sequence was isolated using Bam HI andXba I, and subcloned to the plasmid vector pFastBac™ 1 (GibcoBRL,Gaithersburg, Md.). After subcloning, recombinant baculovirus DNA(hereafter referred to as Bacmid) was generated by transformingpFastBac™ 1 containing the mutein cDNA to the E. coli strain DH10Bac™(GibcoBRL, Gaithersburg, Md.) as described by the manufacturer. Muteinswere expressed in Spodoptera frugiperda (SJ) 9 cells using theBac-to-Bac (GibcoBRL, Gaithersburg, Md.) baculovirus expression system.All insect cell incubations occurred at 28° C. Briefly, 2 ml cultures ofSf 9 cells were transfected with 5 μL of recombinant Bacmid usingCellFECTIN (GibcoBRL, Gaithersburg, Md.). The supernatant was harvested60 hours post-transfection, and used to infect a 100-200 ml culture of1×10⁶ Sf 9 cells/ml in Grace's media (GibcoBRL, Gaithersburg, Md.). Permanufacturer's protocol, the supernatants were harvested 48-60 hrspost-infection by centrifugation at 5000 rpm for 10 minutes in aSorvall® RC-5B centrifuge using a GSA rotor (Dupont Instrument Co.,Willmington, Del.) and assayed for virus titre (typically, >1×10⁸ plaqueforming units/ml was obtained). For protein production, 2-3×10⁶ Sf9cells/ml in 500 mls of SF900 II media (GibcoBRL, Gaithersburg, Md.) wereinfected at a multiplicity of infection between 4-10 and the supernatantwas harvested 60-72 hrs post-infection by centrifugation at 5000 rpm for10 minutes in a Sorvall® RC-5B centrifuge using a GSA rotor (DupontInstrument Co., Willmington, Del.) and filtered through a sterile 0.2 μMfilter unit.

Example 2 Purification of muteins

[0123] Anti-human IL-4 monoclonal antibodies C400.1 and C400.17 weregenerated using standard protocols from mice using recombinant humanIL-4 (Genzyme Diagnostics, Cambridge, Mass.) as immunogen, were producedas ascites fluid, purified, and coupled to CNBr-activated Sepharose(Pharmacia, Uppsala, Sweden) as per manufacturer's protocol. Sf9 cellsupernatants generated from infection of Sf 9 cells by recombinantbaculovirus containing the respective IL-4 mutein were loaded onto a 1ml column of IL-4 affinity matrix, washed with 100 mM NaHCO₃, 500 mMNaCl, pH 8.3, washed with water to remove salt, and eluted with 8 columnvolumes of 100 mM Glycine, pH 3.0. Fractions were collected insiliconized vials containing 0.1 volume 1M Tris, pH 8.0. Mutein proteinwas further purified by reverse phase chromatography using aDynamax®-300Å C₁₈ column (Rainin Instrument Co., Woburn, Mass.) with a0-100% gradient of Buffer A to B (Buffer A, water; Buffer B,acetonitrile, 0.1% trifluoroacetic acid). Fractions were evaluated bySDS-PAGE, and mutein containing fractions were lyophilized for storage,and resuspended in sterile phosphate-buffered saline for assays. Muteinso purified was typically a single band as observed by SDS-PAGL (silverstain), and was quantitated by amino acid analysis (accuracy typically>90%).

Example 3 1° T cell proliferation assay

[0124] Primary T cells were obtained from fresh blood from normal donorsand purified by centrifugation using Ficoll-Paque® Plus (Pharmacia,Upsalla, Sweden) essentially as described by Kruse, N., Tony, H. P. andSebald, W. “Conversion of human interleukin-4 into a high affinityantagonist by a single amino acid replacement”, Embo J. 11: 3237-44(1992). The purified peripheral blood mononuclear cells were incubatedfor 7 days with 10 μg/ml phytohemagglutinin (Sigma Chemical Co., St.Louis, Mo.), harvested by centrifugation, and washed in RPMI 1640 media(GibcoBRL, Gaithersburg, Md.). 5×10⁴ activated T cells/well (PHA-blasts)were incubated with varying amounts of IL-4 or mutein in RPMI 1640 mediacontaining 10% fetal bovine serum, 10 mM HEPES, pH 7.5, 2 mML-glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycinsulphate in 96 well plates for 72 hrs at 37° C., pulsed with 1 μCi³H-thymidine (DuPont NEN®, Boston, Mass.)/well for 6 hrs, harvested, andradioactivity was measured in a TopCount™ scintillation counter (PackardInstrument Co., Meriden, Conn.).

Example 4 HUVEC IL-6 secretion assay

[0125] Human umbilical vein endothelial cells (HUVEC) were obtained fromClonetics® Corp. (San Diego, Calif.), and maintained as per supplier'sprotocols. Cells (passage 3 to 6) were harvested by incubation withTrypsin/EDTA, washed, and plated at subconfluent densities in 48-wellplates in EGM® media (Clonetics® Corp., San Diego, Calif.) containingbovine brain extract (BBE; Clonetics® Corp., San Diego, Calif.). Atconfluency (3-4 days at 37° C.), the media was removed and replaced withEGM® media without BBE. 24 hours later, varying concentrations of IL-4or mutein was added to the cells in fresh EGM® without BBE, and allowedto incubate an additional 24 hrs. Supernatants were harvested and theconcentration of IL-6 was analyzed using a human IL-6 ELISA. Theconditions were identical except for antagonist assays, varyingconcentrations of mutein were added to a constant concentration of 100pM IL-4. Briefly, 96-well Immunolon® 2 plates (Dynatech Laboratories,Inc., Chantilly, Va.) were coated with 5 μg/ml anti-human IL-6 MAbCat#1618-01 (Genzyme Diagnostics, Cambridge, Mass.) overnight at 4° C.Human IL-6 standard (Genzyme Diagnostics, Cambridge, Mass.) or sampleswere titrated in duplicate and incubated with the coated plate; afterwashes, secondary antibody rabbit anti-human IL-6 PAb (CaltagLaboratories, South San Francisco, Calif., Cat#PS-37) at a 1:1000dilution was added. The presence of bound rabbit anti-IL-6 PAb wasdetected using alkaline phosphatase-coupled donkey anti-rabbit Ig PAb(Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.,Cat#711-055-152) diluted 1:2000, and developed using pNPP (SigmaChemical Co., St. Louis, Mo., Cat#N2770 or N1891). Absorbance was readat 405 nm using a Vmax™ kinetic microplate reader (Molecular DevicesCorp., Menlo Park, Calif.).

Example 5 Activities of Muteins

[0126] Table 1 summarizes the results of the muteins in the two assaysdescribed above. “EC₅₀, pM” is the effective concentration that producesa 50% maximal response measured in the concentration picomoles/liter.Activity is a function of both potency (EC₅₀) and maximal response(R_(max)). Cell-selective muteins exhibited differential activity ofeither a relative reduction in R_(max) and/or a relative reduction inpotency (increase in EC₅₀) in the HUVEC assay vs. the T cell assay.“R_(max), % wt” is the maximal response measured relative to wild-typeIL-4. By definition, wild-type IL-4 gives 100% response. All muteinswere active in the T cell proliferation assay. Muteins R121D, R121E,R121P, and R121T/E122F/Y124Q were more potent than wild-type IL-4 inthis assay, although mutein R121T/E122F/Y124Q had a reduced maximalresponse. Muteins Y124Q, Y124R, and Y124A/S125A had 2-3-fold increasedEC₅₀ values than wild-type, as well as a reduced maximal response.However, they appear to retain a significant proportion of IL-4 activityon T cells. Muteins R121E, Y124Q and R121T/E122F/Y124Q had no measurableactivity in the HUVEC assay, making them clearly T cell-selective, andthus selective for the IL-4 receptor expressed on T cells(IL-4Rα/IL-2Rγ). These muteins are IL-4 antagonists on endothelial cellsbecause, although they interact normally with IL-4Rα, they do notactivate the complex IL-4Rα/γ-like subunit. The muteins R121P and Y124Rshow activity in the HUVEC assay, but their EC₅₀ values are increasedbetween 50-150-fold, and have reduced maximal responses relative totheir ability to stimulate T cells. Although these two proteins do notappear to be absolutely T cell-selective, they are preferential fortheir activation of the T cell IL-4 receptor over the HUVEC IL-4receptor. TABLE 1 Muteins with preferential activity on T cells vs.endothelial cells 1° T cell proliferation HUVEC, IL-6 secretion MuteinEC₅₀, pM R_(max), % wt EC₅₀, pM R_(max), % wt wildtype IL-4 150 100  20100  R121A 150 100  20 65 R121D 100  40 —  0 R12lE  65 100 —  0 R121F150 100  20 60 R121H 150  80  40 70 R121I 100 100  40 50 R121K 150 100100 75 R121N 150 100  35 50 R121P 100 100 650 45 R121T 150 100  20 75R121W 150 100  80 35 R121E/T13D 100 100 —  0 Y124A 150  50  65 50 Y124Q250  15 200 25 Y124R 750  30 250 25 Y124S 425  15 350 20 Y124T 300  15350 35 Y124A/S125A 600  60 200 30 R121T/E122F/  15  13 —  0 Y124Q

Example 6 Biological response of IL-4 muteins in HUVEC assays

[0127]FIGS. 2A and B are a series of dose-response plots of HUVEC IL-6secretion by the T cell-selective agonists relative to wild-type. IL-4was included as an internal control on each plate used to assay muteinactivity; a representative curve is shown. FIG. 4A is the dose-responsecurve for R121E versus wild-type IL-4. Similarly, FIGS. 4B-F are thedose-response curves for R121P, Y124Q, Y124R, Y124A/S125A, andR121T/E122F/Y124Q, respectively, versus wild-type IL-4. Activities havebeen normalized relative to the IL-4 control responses. Muteins R121E,Y124Q, and R121T/E122F/Y124Q demonstrate no activity in this assay.Muteins R121P and Y124R, though showing partial agonist activity in thisassay, are relatively less potent to wild-type IL-4 than they are in the1° T cell assay. Thus, despite their activity, they still demonstratepreferential activation of the T cell IL-4 receptor.

Example 7 Biological response of IL-4 muteins in 1° T cell assays

[0128]FIGS. 4A and B show dose-response curves of the T cell-selectiveagonist muteins in a representative assay using cells from a normaldonor. IL-4 was included as an internal control on each plate used toassay mutein activity; a representative curve is shown. FIG. 6A is thedose-response curve for R121E versus wild-type IL-4. Similarly, FIGS.6B-F are the dose-response curves for R121P, Y124Q, Y124R, Y124A/S125A,and R121T/E122F/Y124Q, respectively, versus wild-type IL-4. Activitieshave been normalized relative to the IL-4 control responses. MuteinsR121E, R121P, and R121T/E122F/Y124Q are more potent than wild-type IL-4,although R121T/E122F/Y124Q is only a partial agonist in this assay.Although not as potent as wild-type IL-4 in this assay, muteins Y124Q,Y124R, and Y124A/S125A are still effective partial agonists (R_(max)values 60-70% of wild-type). In FIG. 6, activity is relative to the IL-4response seen in the same plate for each mutein. Of particular note aremuteins R121E and Y124Q, which show significant activity in the T cellassay yet no apparent activity in the HUVEC assay. Each of these muteinsis a clear T cell-selective agonist.

Example 8 Antagonism of HUVEC IL-4-induced IL-6 secretion by T cellselective muteins

[0129] T cell-selective IL-4 muteins R121E () and Y124Q (∇), and theIL-4 antagonist R121D/Y124D (O) (FIG. 7), were titrated against aconstant concentration of 100 pM IL-4. The IL-4 antagonist R121D/Y124Dantagonizes IL-4 with a K_(I) of ˜1.5 nM under these conditions. HUVECdo not express IL-2Rγ, but do express the IL-4 receptor γ-like subunit.The substituted residues of R121E and Y124Q are in the D-helix of IL-4and thus only affect interactions of IL-4 with IL-2Rγ (functionalinteraction) and the γ-like subunit (no or non-functional interaction),but do not affect the ability of these muteins to bind to IL-4Rα. Thus,the T cell-selective agonists R121E and Y124Q, by virtue of theirability to selectively interact with IL-2Rγ on T cells without promotingactivation of the γ-like receptor subunit on endothelial cells, are ableto compete IL-4-induced responses on these endothelial cells (K_(I)˜0.8-1 nM under these conditions). Such antagonism by T cell-selectiveIL-4 muteins may antagonize the effects of endogenously produced IL-4 onendothelial cells during T cell-directed therapy with said muteins.

Examples 9 Biological response of IL-4 mutein R121D

[0130] The IL-4 mutein R121D (Arg-121 substituted with Asp) wasgenerated as described and assayed in the 1° T cell and HUVEC assays.Referring to FIGS. 8A and 8B, data is shown for IL-4 (O) and R121D ()in both the T cell (FIG. 8A) and the HUVEC (FIG. 8B) assays. In the Tcell assay, R121D exhibited an EC₅₀ of ˜100 pM and an R_(max) value of˜60% relative to wild-type IL-4. It was inactive in the HUVEC assay(EC₅₀=; R_(max)=O). These results demonstrate that the singlesubstitution of Arg-121 of human IL-4 with Asp yields a protein that hasselective activity on T cells, and lacks any apparent activity onendothelial cells. Although the mutein R121D is a partial agonist in theT cell assay, it is more potent than wild-type IL-4. However, likemutein R121E, R121D exhibits no activity in the HUVEC assay,demonstrating that it is a clear T cell-selective agonist.

Example 10 Biological activity of IL-4 mutein T13D/R121E

[0131] The IL-4 mutein T13D/R121E (Thr-13 substituted with Asp togetherwith Arg-121 substituted with Glu) was generated as described andassayed in the 1° T cell and HUVEC assays. Specifically with referenceto FIGS. 9A-C, in the T cell assay (Panel A), T13D/R121E (▴) exhibitedan EC₅₀ of ˜100 pM, about 2-3-fold better than IL-4 (O) or R121E (Δ),and an R_(max) value of 100% relative to IL-4. In HUVEC antagonistassays (Panel B), T13D/R121E (▴) was ˜27-fold more potent an antagonistof IL-4 activity than R121E (Δ), exhibiting an IC₅₀ of ˜2.2 nM vs. ˜60nM, respectively. The substitution of Thr-13 to Asp increases thepotency of the T13D/R121E relative to R121E (both as an agonist in the Tcell assay, and as an antagonist in the HUVEC assay), while thesubstitution Arg-121 to Glu confers T cell-selective activity toT13D/R121E (full agonist in the T cell assay, IL-4 antagonist in theHUVEC assay). In the HUVEC assay shown (Panel C), T13D/R121E (▴)exhibited nominal activity comparable to that seen with R121E (Δ). Inthis assay, measurement of monocyte chemoattractant protein 1 (MCP-1) inthe media by ELISA was used instead of IL-6 to measure activity.

Example 11 Chimpanzee Toxicology and Immunopharmacology Study

[0132] Wild-type IL-4 (“wtIL-4”) and IL-4[T13D/R121E] (“IL-4SA”) werecompared in a battery of tests in a two-phase study.

OBJECTIVE

[0133] The primary objective of this study was to determine whetherIL-4SA has reduced toxicity relative to wtIL-4. A secondary objectivewas to determine whether the immunopharmacologic activity of wtIL-4 issimilar to that of IL-4SA. Since very limited information was availableprior to the start of the study regarding the in vivo effects of IL-4SA,both toxicity and immunomodulatory responses were assessed using a largenumber of endpoints.

METHODS

[0134] IL-4SA was originally chosen for development on the basis of itsreceptor selectivity. Since chimpanzee was determined to be the onlyspecies in which IL-4SA was highly specific for the same IL-4 receptorsubtypes as in humans, the chimpanzee was considered to be the onlyviable preclinical model for accurately predicting IL-4SA safety andpharmacology in man.

[0135] The safety pharmacology study was performed according to the FDAguidelines for GLP. The materials and methods are largely described indetail in Protocol 98-15 (on file with the Bayer Berkeley PreclinicalDepartment). The test articles were IL-4SA mutein T13D/R121E (lotnumbers 97253-22 and 98251-96-14) and wtIL-4 (lot numbers 96241-41 and723-1/95). The in-life portion of the study took place at New IberiaResearch Center (“NIRC,” New Iberia, La.) and involved 16 young adult toadult male and female chimpanzees with body weights that ranged fromabout 35 to 80 kg.

[0136] Table 2 provides an overview of the experimental design andindicates that the chimpanzees received once daily subcutaneous (s.c.)doses of test article for either 14 days (in Phase I) or 21 days (inPhase II). Following the cessation of dosing, the animals were continuedon study for an additional 8-day period to determine if the effectscaused by the test articles were reversible. Throughout the study, theanimals were observed twice daily for any signs of reduced foodconsumption, behavioral abnormalities and general health. TABLE 2Overview of experimental design. In each of two portions of the study(Phase I and Phase II), there were four dose groups. The animals weredosed subcutaneously, once daily (between 8:00 and 10:00 a.m.), eitherfor 14 days (in Phase I) or 21 days (in Phase II), then observed for anadditional 8 days (the ‘recovery phase’ of each experiment). Thetreatment groups were as follows: Group Number of Number Animals TestArticle Dose (μg/kg) Phase I 1 2 wtIL-4 10 2 2 Vehicle NA 3 2 IL-4SA100  4 2 IL-4SA 10 Phase II 1 2 wtIL-4 30 2 2 Vehicle NA 3 2 IL-4SA 300 4 2 IL-4SA 30

[0137] Whole blood, serum and plasma samples were analyzed by NIRC for ahost of toxicity and immunomodulatory markers. The toxicity markersincluded a standard serum chemistry panel, a hematology panel coveringcomplete blood counts, differentials and platelet counts, andcoagulation profiling using assays for prothrombin time, activatedpartial prothrombin time, and fibrinogen. To assess immunomodulation,NIRC used fluorescence activated cell sorting (FACS) to follow a widerange of cell-associated proteins. These included markers that allowedfor demographic characterizations of the cellular populations (e.g., thepercentage of total lymophocytes that were B cells) and markers thatreflected phenotypic changes in immune cells (e.g., enhanced expressionof cell-associated CD23).

[0138] In addition to the assays performed at NIRC, the Bayer BerkeleyDepartment of Preclinical Development and PPD Pharmaco (Richmond, Va.)analyzed plasma and serum samples for a wide range of responses that aretypical of prolonged exposure to exogenous IL-4. These includedantibodies against the IL-4 proteins (wtIL-4 and IL-4SA), test articleconcentrations in plasma before and after dosing, soluble IL-4 receptor(sIL-4R), soluble CD23, IgE, MCP-1, and endogenous IL-4.

RESULTS/DISCUSSION

[0139] A. Toxicity Endpoints

[0140] Wild type IL-4 was used in the study as a reference for comparingboth the adverse and immunododulatory effects of IL-4SA. In humans, i.v.bolus administration of wtIL-4 at 20 μg/kg t.i.d. has been reported toresult in reversible renal dysfunction, upper gastrointestinal tractbleeding, capillary leak syndrome, diarrhea, and carditis. Given thepotential for this molecule to have severe toxicity, its dosage in thesafety pharmacology study with chimpanzees was carefully selected inclose consideration of published articles describing IL-4 toxicity inprimates. The established dose for wtIL-4 in chimpanzees in the Phase Iexperiment, 10 μg/kg/day qd for 14 days was anticipated to result inclinically significant, but mild toxicity, with completely reversibleclinical pathology. However, no clinically significant toxicity wasstatistically significant at this dose level, so the Phase II dose wasincreased to 30 μg/kg/day for wtIL-4.

[0141] Although none of the changes in endpoints were of a magnitudethat would constitute clinical significance, the coagulation parameters,fibrinogen and activated partial prothrombin time (“APTT”), showed cleartreatment-related trends. Thus the greatest impact was seen with wtIL-4at 10 μg/kg and no discernible effect was observed with IL-4SA at either10 or 100 μg/kg (FIGS. 10A-10D). FIGS. 10A through 10D show the changein Day 1 levels of fibrinogen and APTT as a function of exposure to testarticle. FIGS. 10A and 10B both show a deep reduction (50% or more) infibrinogen production at the 30 μg/kg/day wtIL-4 dose, but no suchreduction for either 30 or 300 μg/kg/day IL-4SA. FIGS. 10C and 10D alsoshow increases in APTT at the 30 μg/kg/day wtIL-4 dose level ofapproximately 20%, while the 30 μg/kg/day IL-4SA dose shows little or noeffect.

[0142] In contrast to the Phase I experiment, in which the twice-dailyobservations of the chimpanzees revealed no outwardly apparent toxicity,clinically significant events were observed in Phase II. These involvedtransient edema of the neck and face on day 14 with two animals, onedosed with 30 μg/kg wtIL-4 and the other with 300 μg/kg wtIL-4. The sameanimal from the 30 μg/kg wtIL-4 treatment later presented scrotal edemaon day 20, which exacerbated on day 21 to include abdominal edema. Theedema in this animal was sufficiently severe to warrant discontinuationof treatment with wtIL-4, but no action was ultimately taken since day21 was the final day of dosing as prescribed by the protocol.

[0143] To rule out the possibility that differential effects observed inthe toxicity wtIL-4 and IL-4SA were attributed to ‘neutralization’ byantibodies against either of these proteins, assays were performed forIgG against both proteins. In the Phase I experiment, these assaysshowed that antibodies were not raised against either protein in any ofthe chimpanzees. In the Phase II experiment, antibody formation wasconfirmed in only two animals: one dosed with 300 μg/kg IL-4SA and oneadministered 30 μg/kg IL-4SA. However, these antibodies appeared fairlylate in the study (starting on day 17), and therefore seem unlikely toaccount for a reduction in toxicity with IL-4SA during the first twoweeks of the Phase II experiment.

[0144] B. Immunopharmacological Endpoints

[0145] To assess immunopharmacologic activity, a wide range of responseswas followed that are typical of prolonged exposure to exogenous IL-4.These included IL-4R, CD4 and CD23. The Phase II experiment showed that30 ug/kg/day wtIL-4, 30 ug/kg/day IL-4SA and 300 ug/kg/day IL-4SA wereroughly equipotent at affecting the percentages of circulatinglymphocytes staining positive for CD23 (FIG. 11A) and CD4 plus IL-4R(FIG. 11B). However, the total number of circulating lymphocytesstaining positive for CD4 alone showed a dose response relationship inwhich peak levels were greatest with 300 ug/kg/day IL-4SA (FIGS. 12A,12B and 12C). Combined data from the Phase I and II experiments suggestthat 30 ug/kg wtIL-4 was approximately equipotent to 30 ug/kg IL-4SA ataffecting the total number of circulating CD4 positive lymphocytes (FIG.12C). These observations surrounding CD4 positive cells are noteworthyin that they indicate that IL-4SA is similar to wtIL-4 in affectingcertain populations of T cells. Overall, the immunopharmacology datasuggest that IL-4SA possesses many of the immunomodulatory properties ofwtIL-4. These data serve to establish that the reduced toxicity observedwith IL-4SA relative to wtIL-4 is not attributed to biologicalinertness. In conclusion, the experiments with chimpanzees showed thatIL-4SA is significantly less toxic than wtIL-4, but has comparablepotency in affecting certain populations of lymphocytes.

Example 12 Treatment of multiple sclerosis with IL-4 selective agonist

[0146] The use of an animal model as a predictor for pharmacologicalutility in humans is a well-accepted research tool. Initial testing ofthe IL-4 selective agonist for multiple sclerosis (MS) is conducted in amarmoset model using recombinant human IL-4 selective agonist protein.These studies are conducted to examine the effect of prophylactic andtherapeutic treatment on disease induction and severity for both theacute symptomology as well as chronic relapsing-remitting disease.

[0147] Experimental autoimmune encephalomyelitis (EAE) is a CD4+ Tcell-mediated autoimmune, inflammatory disease of the central nervoussystem. Induction of EAE is induced in marmosets (C. jacchus) weighing300 to 400 gm by immunization with 200 mg of fresh-frozen postmortemhuman brain white matter homogenate (BH) emulsified with completeFreund's adjuvant (CFA) containing 3 mg/ml of killed Mycobacteriumtuberculosis as described in Massacesi et al., Ann. Neurol., 37:519(1995). On the day of immunization and again 2 days later, 10¹⁰inactivated Bordetella pertussis organisms are diluted in 10 ml ofsaline solution and administered intravenously.

[0148] EAE is assessed by clinical and pathological criteria. Astandardized scoring system is employed to record the severity ofclinical disease: 0=normal neurological findings; 1=lethargy, anorexia,weight loss; 2=ataxia, and either paraparesis/monoparesis, sensory loss,or brainstem syndrome including gaze palsy, or blindness; 3=paraplegiaor hemiplegia; 4=quadriplegia.

[0149] Magnetic resonance imaging (MRI) has been shown to be a usefultechnique to characterize early as well as late immune mediated lesionsof MS (Stewart et al., Brain, 114:1069 (1991). MRI is used to evaluateanimals after immunization to monitor progression of disease over time.MRI data is collected on a Picker International NMR Cryogenic ‘2000’system, operating at a field strength of 0.15 Tesla; a receiver coilwith an aperture of 15 cm to obtain the images. Multislice spin-echo andinversion-recovery pulse sequences are employed. Echo-delays times ofeither 40 and 60 ms, or 40 and 80 ms are used in the spin-echosequences. In the inversion-recovery sequences the 180-90 interpulsedelay is 400 ms.

[0150] Marmosets are anesthetized with ketamine hydrochloride and placedin the scanner using a laser available for patient alignment such thatthe inner canthi of the eyes are aligned perpendicular to the directionof the static magnetic field. Animals are scanned before immunizationand then daily from day 9 after immunization. Prior to scanning eachday, animals are checked for signs of neurological impairment.

[0151] Animals are sacrificed at different times after immunization. TheCNS is removed and fixed in 10% formalin. Paraffin sections of brain andspinal cord are prepared and stained with hematoxylin and eosin. Eachcoronal brain section or horizontal spinal cord section is analyzed forhistopathological findings of inflammation and demyelination accordingto an arbitrary scale: inflammation; 0=no inflammation present, +=rareperivascular cuffs/average whole section; ++=moderate numbers ofperivascular cuffs/section; may have meningeal inflammation;+++=widespread perivascular cuffing and parenchymal infiltration byinflammatory cells. Demyelination score; 0=no demyelination present;+=rare foci of demyelination; ++=moderate demyelination; +++=extensivedemyelination with large confluent lesions.

[0152] For pretreatment studies on acute disease pathology, test drug isadministered subcutaneously at a dosage range between 1 and 500 ug/kgfollowing a dosing regimen of 1 administration per day to 1administration per week prior to the onset of disease symptoms. Fortherapeutic intervention in existing disease, test article isadministered subcutaneously at a dose range between 1 and 500 ug/kgfollowing an extended dosing regimen of 1 treatment per day to 1treatment per week over the course of several months.

Example 13 Treatment of rheumatoid arthritis

[0153] Rheumatoid arthritis (RA) is a debilitating inflammatory diseasein which chronic activation of resident and infiltrating synovial cellscauses destruction of cartilage and bone and leads to fibrosis and lossof function. Cytokines released from activated T cells are thought toplay a role in the maintenance of the chronic inflammatory reaction.

[0154] RA is induced in DBA/1 mice using type II collagen as describedby Joosten et al., Arthritis & Rheumatism; 39:797 (1996). Collageninduced arthritis (CIA) is induced by immunizing mice via intradermalinjection at the base of the tail with 100 ul of emulsion containing 100ug of collagen. On day 21, animals are given a intraperitoneal boosterinjection of type II collagen (100 ug) dissolved in phosphate bufferedsaline (PBS).

[0155] Assessment of CIA is performed by examining the mice visually forthe appearance of arthritis in the peripheral joints and scores forarthritis severity are assigned. Mice are considered to have arthritiswhen significant changes in redness and/or swelling is noted in thedigits or in other parts of a minimum of 2 paws.

[0156] Clinical severity of arthritis is scored on a scale of 0-2 foreach paw according to changes in redness and swelling (0=no change,0.5=significant, 1.0=moderate, 1.5=marked and 2.0=severe maximalswelling and redness. Scoring is assessed by at least two blindedobservers.

[0157] At the end of the study, some of the animals are sacrificed andpaw and joint tissue is obtained for pathological and histopathologyexamination. The tissue is processed for immunohistochemical staining(frozen sections) or fixed and embedded in paraffin, sectioned andstained with H&E for analysis of cellular infiltration.

[0158] Evaluation of a murine analog of the IL-4 selective agonist ofthe present invention in the CIA model is performed with the use of amurine equivalent protein molecule. One of ordinary skill in the art iscapable of comparing the murine IL-4 structure with the human IL-4structure, generating parallel murine IL-4 muteins, and making anynecessary adjustments based on responses in in vitro assays utilizingcell lines expressing either IL-4Rα/IL-2Rγ or IL-4Rα/γ-like subunit in amanner analogous to that used for human IL-4 muteins with T cells andHUVEC. Animals are dosed one day prior to the booster administration ofcollagen and kept on a dosing regimen ranging between once a day to oncea week for the duration of the study (40+ days). Animals are dosed witha range of concentrations of IL-4 selective agonist ranging between 1 to100 ug/kg.

Example 14 Treatment of insulin dependent diabetes mellitus (IDDM)

[0159] There is some evidence in the literature of Th1 cell involvementin IDDM in humans and animal models of human disease. Nonobese diabetic(NOD) mice are utilized to examine the efficacy of a murine IL-4equivalent of IL-4 selective agonist in treating IDDM. One of ordinaryskill in the art is capable of comparing the murine IL-4 structure withthe human IL-4 structure, generating parallel murine IL-4 muteins, andmaking any necessary adjustments based on responses in in vitro assaysutilizing cell lines expressing either IL-4Rα/IL-2Rγ or IL-4Rα/γ-likesubunit in a manner analogous to that used for human IL-4 muteins with Tcells and HUVEC. Prediabetic NOD mice (approximately 7 wks) exhibit aproliferative unresponsiveness in vitro after T cell stimulation. Thetiming of this unresponsiveness is not related to insulitis and persistsuntil the onset of diabetes which occurs at 24 wks of age.

[0160] Evaluation of the IL-4 selective agonist in NOD mice is conductedsimilar to studies reported by Rapoport et al., J. Exp Med; 178; p. 87(1993). NOD mice are injected with test material at approximately 3 wksof age following a dosing regimen of once daily treatment or once a weektreatment over the course of 12 weeks until the mice are 15 wks old. Acontrol group of animals will receive treatment with a inert proteinequivalent.

[0161] Mice will we tested for glycosuria using Tes-Tape and diagnosedfor diabetes as determined by being glycosuria for at least twoconsecutive weeks. At the end of 52 wks, animals are sacrificed toobtain various organs and tissue for pathology evaluation. Tissue fromthe pancreas, submandibular salivary glands and kidney from each mouseis fixed and embedded in paraffin, sectioned and stained. Aldehydefuchsin staining of pancreas sections is used to examine the extent towhich insulitic infiltrates have reduced the mass of granulated β cells.Splenic leukocytes are counted by FACScan analyses using anti-Thy-1.2,anti-CD4 and anti-CD8 mabs in ascites as described by Zipris et al., J.Immunol 146; p. 3763 (1991).

[0162] Other embodiments of the invention will become apparent to one ofskill in the art. This invention teaches how to obtain muteins notspecifically described herein but which have T cell activating abilityand reduced endothelial cell activating ability, and thereby thosemuteins come within the spirit and scope of the invention. The conceptand experimental approach described herein should be applicable to othercytokines utilizing heterologous multimeric receptor systems, inparticular IL-2 and related cytokines (e.g., IL-7, IL-9 and IL-15),IL-10, interferon α, and interferon γ.

SEQUENCES

[0163] The following sequences are contained within this application:

[0164] SEQ ID NO: 1: hIL-4 (amino acid)

[0165] SEQ ID NO: 2: hIL-4 (amino acid, cDNA)

[0166] SEQ ID NO: 3: R121A (amino acid, cDNA)

[0167] SEQ ID NO: 4: R121D (amino acid, cDNA)

[0168] SEQ ID NO: 5: R121E (amino acid, cDNA)

[0169] SEQ ID NO: 6: R121F (amino acid, cDNA)

[0170] SEQ ID NO: 7: R121H (amino acid, cDNA)

[0171] SEQ ID NO: 8: R121I (amino acid, cDNA)

[0172] SEQ ID NO: 9: R121K (amino acid, cDNA)

[0173] SEQ ID NO: 10: R121N (amino acid, DNA)

[0174] SEQ ID NO: 11: R121P (amino acid, cDNA)

[0175] SEQ ID NO: 12: R121T (amino acid, cDNA)

[0176] SEQ ID NO: 13: R121W (amino acid, cDNA)

[0177] SEQ ID NO: 14: Y124A (amino acid, cDNA)

[0178] SEQ ID NO: 15: Y124Q (amino acid, cDNA)

[0179] SEQ ID NO: 16: Y124R (amino acid, cDNA)

[0180] SEQ ID NO: 17: Y121S (amino acid, cDNA)

[0181] SEQ ID NO: 18: R121T (amino acid, cDNA)

[0182] SEQ ID NO: 19: Y124A/S125A (amino acid, cDNA)

[0183] SEQ ID NO: 20: T13D/R121E (amino acid, cDNA)

[0184] SEQ ID NO: 21: R121T/E122F/Y124Q (amino acid, cDNA)

[0185] SEQ ID NO: 22: 5′ PCR Primer, IL-4

[0186] SEQ ID NO: 23: 3′ PCR Primer, IL-4

[0187] SEQ ID NO: 24: Mutagenesis Primer for R121A

[0188] SEQ ID NO: 25: Mutagenesis Primer for R121D

[0189] SEQ ID NO: 26: Mutagenesis Primer for R121E

[0190] SEQ ID NO: 27: Mutagenesis Primer for R121F

[0191] SEQ ID NO: 28: Mutagenesis Primer for R121H

[0192] SEQ ID NO: 29: Mutagenesis Primer for R121I

[0193] SEQ ID NO: 30: Mutagenesis Primer for R121K

[0194] SEQ ID NO: 31: Mutagenesis Primer for R121N

[0195] SEQ ID NO: 32: Mutagenesis Primer for R121P

[0196] SEQ ID NO: 33: Mutagenesis Primer for R121T

[0197] SEQ ID NO: 34: Mutagenesis Primer for R121W

[0198] SEQ ID NO: 35: Mutagenesis Primer for Y124A

[0199] SEQ ID NO: 36: Mutagenesis Primer for Y124Q

[0200] SEQ ID NO: 37: Mutagenesis Primer for Y124R

[0201] SEQ ID NO: 38: Mutagenesis Primer for Y124S

[0202] SEQ ID NO: 39: Mutagenesis Primer for Y124T

[0203] SEQ ID NO: 40: Mutagenesis Primer for Y124A/S125A

[0204] SEQ ID NO: 41: Mutagenesis Primer for T13D

[0205] SEQ ID NO: 42: Mutagenesis Primer for R121T/E122F/Y124Q

[0206] Note: for the T13D/R121E mutein, the primers SEQ ID NOs: 26 and41 are used.

1 42 129 amino acid single linear protein human Interleukin-4 protein nono 1 His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn 1 5 1015 Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr 20 25 30Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe 35 40 45 CysArg Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu 50 55 60 Lys AspThr Arg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg 65 70 75 His Lys GlnLeu Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu 80 85 90 Trp Gly Leu AlaGly Leu Asn Ser Cys Pro Val Lys Glu Ala Asn 95 100 105 Gln Ser Thr LeuGlu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met 110 115 120 Arg Glu Lys TyrSer Lys Cys Ser Ser 125 462 nucleic acid single linear cDNA human IL-4protein no no 2 ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTGCTA 45 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 510 15 GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 AlaCys Ala Gly Asn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAGGAG ATC ATC AAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu IleIle Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAGTTG ACC GTA ACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu ThrVal Thr Asp Ile Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACCTTC TGC AGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe CysArg Ala Ala Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GACACT CGC TGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr ArgCys Leu 80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATCCGA 315 Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95100 105 TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn SerCys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTGGAA AGG CTA AAG ACG ATC ATG AGA GAG AAA TAT TCA AAG 450 Phe Leu Glu ArgLeu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG462 Cys Ser Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121Ano no 3 ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala CysAla Gly Asn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAGATC ATC AAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile IleLys Thr Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTGACC GTA ACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr ValThr Asp Ile Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTCTGC AGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys ArgAla Ala Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACTCGC TGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg CysLeu 80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA315 Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100105 TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 PheLeu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AATTCC TGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser CysPro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAAAGG CTA AAG ACG ATC ATG GCT GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg LeuLys Thr Ile Met Ala Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462Cys Ser Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121D nono 4 ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 MetGly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCATGT GCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys AlaGly Asn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATCATC AAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile LysThr Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACCGTA ACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val ThrAsp Ile Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGCAGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg AlaAla Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGCTGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG GAC GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Asp Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121E no no 5ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG GAA GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Glu Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121F no no 6ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG TTT GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Phe Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121H no no 7ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG CAC GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met His Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121I no no 8ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG ATA GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Ile Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121K no no 9ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AAA GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Lys Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121N no no 10ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AAC GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Asn Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121P no no 11ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG CCA GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Pro Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121T no no 12ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG ACT GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Thr Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/R121W no no 13ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG TGG GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Trp Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA hIL-4/Y124A no no 14ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG GCA GAG AAA GCA TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Ala Glu Lys Ala Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA IL-4/Y124Q no no 15ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AGA GAG AAA CAA TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Arg Glu Lys Gln Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA IL-4/Y124R no no 16ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AGA GAG AAA CGA TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Arg Glu Lys Arg Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA IL-4/Y124S no no 17ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AGA GAG AAA TCA TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Arg Glu Lys Ser Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA IL-4/Y124T no no 18ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met GlyLeu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGTGCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala GlyAsn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATC ATCAAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTAACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr AspIle Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AGGGCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala AlaThr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGCCTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 8085 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AGA GAG AAA ACA TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Arg Glu Lys Thr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA IL-4/Y124A/S125A nono 19 ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 MetGly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCATGT GCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys AlaGly Asn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATCATC AAA ACT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile LysThr Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACCGTA ACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val ThrAsp Ile Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGCAGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg AlaAla Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGCTGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG AGA GAG AAA GCT GCT AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Arg Glu Lys Ala Ala Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNA IL-4/T13D/R121E nono 20 ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 MetGly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCATGT GCC GGC AAC TTT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys AlaGly Asn Phe Val His Gly His Lys Cys Asp Ile Thr 20 25 30 TTA CAG GAG ATCATC AAA GAT TTG AAC AGC CTC ACA GAG CAG AAG 135 Leu Gln Glu Ile Ile LysAsp Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACCGTA ACA GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val ThrAsp Ile Phe Ala Ala Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TTC TGCAGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg AlaAla Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGCTGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg 95 100 105TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGC CTG GCG GGC TTG 360 Phe LeuLys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 110 115 120 AAT TCCTGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys ProVal Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGGCTA AAG ACG ATC ATG GAA GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu LysThr Ile Met Glu Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG 462 CysSer Ser End 153 462 nucleic acid single linear cDNAhIL-4/R121T/E122F/Y124Q no no 21 ATG GGT CTC ACC TCC CAA CTG CTT CCC CCTCTG TTC TTC CTG CTA 45 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu PhePhe Leu Leu 1 5 10 15 GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AAG TGCGAT ATC ACC 90 Ala Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp IleThr 20 25 30 TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CTC ACA GAG CAG AAG135 Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC ATC TTT GCT GCC TCC 180 Thr LeuCys Thr Glu Leu Thr Val Thr Asp Ile Phe Ala Ala Ser 50 55 60 AAG AAC ACAACT GAG AAG GAA ACC TTC TGC AGG GCT GCG ACT GTG 225 Lys Asn Thr Thr GluLys Glu Thr Phe Cys Arg Ala Ala Thr Val 65 70 75 CTC CGG CAG TTC TAC AGCCAC CAT GAG AAG GAC ACT CGC TGC CTG 270 Leu Arg Gln Phe Tyr Ser His HisGlu Lys Asp Thr Arg Cys Leu 80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGGCAC AAG CAG CTG ATC CGA 315 Gly Ala Thr Ala Gln Gln Phe His Arg His LysGln Leu Ile Arg 95 100 105 TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GGCCTG GCG GGC TTG 360 Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu AlaGly Leu 110 115 120 AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTGGAA AAC 405 Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn125 130 135 TTC TTG GAA AGG CTA AAG ACG ATC ATG ACC TTC AAA CAG TCA AAG450 Phe Leu Glu Arg Leu Lys Thr Ile Met Thr Phe Lys Gln Ser Lys 140 145150 TGT TCG AGC TAG 462 Cys Ser Ser End 153 24 nucleic acid singlelinear cDNA 5′ PCR Primer, IL-4 no no 22 CGCGGATCCA TGGGTCTCAC CTCC 2429 nucleic acid single linear cDNA 3′ PCR Primer, IL-4 no no 23CGCTCTAGAC TAGCTCGAAC ACTTTGAAT 29 28 nucleic acid single linear cDNAMutagenesis Primer, IL-4/R121A no no 24 CTAAAGACGA TCATGGCTGA GAAATATT28 30 nucleic acid single linear cDNA Mutagenesis Primer, IL-4/R121D nono 25 GCTAAAGACG ATCATGGACG AGAAATATTC 30 30 nucleic acid single linearcDNA Mutagenesis Primer, IL-4/R121E no no 26 GCTAAAGACG ATCATGGAAGAGAAATATTC 30 28 nucleic acid single linear cDNA Mutagenesis Primer,IL-4/R121F no no 27 CTAAAGACGA TCATGTTTGA GAAATATT 28 28 nucleic acidsingle linear cDNA Mutagenesis Primer, IL-4/R121H no no 28 CTAAAGACGATCATGCACGA GAAATATT 28 28 nucleic acid single linear cDNA MutagenesisPrimer, IL-4/R121I no no 29 CTAAAGACGA TCATGATAGA GAAATATT 28 28 nucleicacid single linear cDNA Mutagenesis Primer, IL-4/R121K no no 30CTAAAGACGA TCATGAAAGA GAAATATT 28 28 nucleic acid single linear cDNAMutagenesis Primer, IL-4/R121N no no 31 CTAAAGACGA TCATGAACGA GAAATATT28 30 nucleic acid single linear cDNA Mutagenesis Primer, IL-4/R121P nono 32 GCTAAAGACG ATCATGCCAG AGAAATATTC 30 28 nucleic acid single linearcDNA Mutagenesis Primer, IL-4/R121T no no 33 CTAAAGACGA TCATGACTGAGAAATATT 28 28 nucleic acid single linear cDNA Mutagenesis Primer,IL-4/R121W no no 34 CTAAAGACGA TCATGTGGGA GAAATATT 28 28 nucleic acidsingle linear cDNA Mutagenesis Primer, IL-4/Y124A no no 35 ATCATGAGAGAGAAAGCATC AAAGTGTT 28 28 nucleic acid single linear cDNA MutagenesisPrimer, IL-4/Y124Q no no 36 ATCATGAGAG AGAAACAATC AAAGTGTT 28 28 nucleicacid single linear cDNA Mutagenesis Primer, IL-4/Y124R no no 37ATCATGAGAG AGAAACGATC AAAGTGTT 28 28 nucleic acid single linear cDNAMutagenesis Primer, IL-4/Y124S no no 38 ATCATGAGAG AGAAATCATC AAAGTGTT28 28 nucleic acid single linear cDNA Mutagenesis Primer, IL-4/Y124T nono 39 ATCATGAGAG AGAAAACATC AAAGTGTT 28 33 nucleic acid single linearcDNA Mutagenesis Primer, IL-4/Y124A/S125A no no 40 CGATCATGAG AGAGAAAGCTGCTAAGTGTT CGA 33 28 nucleic acid single linear cDNA Mutagenesis Primer,IL-4/T13D T13D no no 41 CAGGAGATCA TCAAAGATTT GAACAGCC 28 34 nucleicacid single linear cDNA Mutagenesis Primer, IL-4/R121T/E122F/Y124Q no no42 GCTAAAGACG ATCATGACCT TCAAACAGTC AAAG 34

We claim:
 1. A polypeptide comprising a human IL-4 mutein numbered inaccordance with wild-type IL-4, said mutein having the substitutionR121E, wherein said substitution substantially preserves native T cellactivating ability but substantially reduces endothelial cell activatingability on the resulting IL-4 mutein, relative to wild-type.
 2. Apolypeptide comprising a human IL-4 mutein numbered in accordance withwild-type IL-4, said mutein having at least both substitutions T13D andR121E, wherein said substitutions substantially preserve native T cellactivating ability but substantially reduce endothelial cell activatingability on the resulting IL-4 mutein, relative to wild-type.
 3. Apharmaceutical composition comprising the human IL-4 mutein of claim 1in combination with a pharmaceutically acceptable carrier.
 4. Apharmaceutical composition comprising the human IL-4 mutein of claim 2in combination with a pharmaceutically acceptable carrier.
 5. Apolynucleotide molecule comprising a polynucleic acid encoding the humanIL-4 mutein of claim 1 and degenerate variants thereof.
 6. Apolynucleotide molecule comprising a polynucleic acid encoding the humanIL-4 mutein of claim 2 and degenerate variants thereof.
 7. A host celltransformed with the polynucleotide of claim
 5. 8. A host celltransformed with the polynucleotide of claim
 6. 9. A method of treatinga patient afflicted with an IL-4 treatable condition by administering atherapeutically effective amount of a human IL-4 mutein of claim 1 or 2.10. The method of claim 9 wherein said IL-4 treatable condition is anautoimmune disorder.
 11. The method of claim 10 wherein said autoimmunecondition is Multiple Sclerosis.
 12. The method of claim 10 wherein saidautoimmune condition is Rheumatoid Arthritis.
 13. The method of claim 10wherein said autoimmune condition is Insulin Dependent Diabetes Melitus.14. The method of claim 10 wherein said autoimmune condition is SystemicLupus Erythematosus.
 15. The method of claim 9 wherein said IL-4treatable condition is an infectious disease.
 16. The method of claim 15wherein said infectious disease is Lyme Disease.
 17. The method of claim9 wherein said IL-4 treatable condition is a Th1-polarized disease. 18.The method of claim 17 wherein said Th1 -polarized disease is Psoriasis.19. The method of claim 9 wherein said IL-4 treatable condition is acancer.
 20. The method of claim 19 wherein said cancer is selected fromthe group consisting of acute lymphoblastic leukemia, and non-HodgkinsLymphoma.
 21. The method of claim 9 wherein said IL-4 treatablecondition is a cartilagenous disorder.
 22. The method of claim 21wherein said cartilagenous disorder is osteoarthritis.